A set of simple stopping criteria is presented, which improve the efficiency of iterative root finding by terminating the iterations immediately when no further improvement of the roots is possible. The criteria use only the function evaluations already needed by the root finding procedure to which they are applied. The improved efficiency is achieved by formulating the stopping criteria in terms of fractional significant digits. Test results show that the new stopping criteria reduce the iteration work load by about one-third compared with the most efficient stopping criteria currently available. This is achieved without compromising the accuracy of the extracted roots. The drawback of such stopping criteria is that they shift the responsibility for providing accurate results from the program developer to the user. Also, functions exist for which such stopping criteria fail  those that rely on the user to specify a tolerance within which the roots are needed and 2) those that seek to terminate the iterations automatically when an iterate has been reached whose accuracy cannot be improved. Both categories are widely used  stopping criteria already exist. They are reviewed in §3. The new stopping criteria are then presented in §§4, 5 and 6. Finally, the new and the old criteria are compared in §7 in terms of efficiency and accuracy.3.3.1zi is declared a root ifA(zi) and B(zi) are both equal to p(zi) but with p(zi) calculated in different ways: A(zi)=p(zi) with p(zi) evaluated as usual by Horner's method. B(zi) must be written as B(zi)=D(zi)−C(zi), where D(z)=zp′(z) and C(z)=zp′(z)−p(z). Both polynomials D(z) and C(z) must be reduced analytically to their simplest form and evaluated separately by Horner's method before B(zi)=D(zi)−C(zi) is calculated. Analytical reduction results inA(zi) and B(zi) approach zero, |A(zi)−B(zi)| will initially be smaller than either |A(zi)| or |B(zi)|, thus preventing the stopping criterion from being satisfied. But, as |A(zi)| and |B(zi)| grow smaller, round-off errors will dominate |A(zi)−B(zi)| before they dominate |A(zi)| and |B(zi)|, thus providing an opportunity for the stopping criterion to be satisfied when |A(zi)−B(zi)| has lost all its significant digits.Igarashi  providesp(zi), p′ (zi) and p′′(zi) are needed by the iteration procedure itself, and D(zi) and C(zi) are needed by the stopping criterion. Thus, Igarashi's stopping criterion adds two-thirds to the work load per iteration. The criterion is included in the comparisons reported in §7.If a third order iteration procedure, such as Laguerre's method (e.g. Orchard ), is use3.2f(z): iterate zi is declared a root ifA(zi) and B(zi) are both equal to f(zi) but with f(zi) calculated in different ways: A(zi)=f(zi) with f(zi) in its standard analytical formulation. B(zi) must be written as B(zi)=D(zi)−C(zi), where D(z)=zf ′(z) and C(z)=zf ′(z)−f(z). Both functions D(z) and C(z) must be reduced analytically to their simplest form before B(zi)=D(zi)−C(zi) is calculated. W must be equal to either 1.0 or 0.5 if f(z) is algebraic, and W must be equal to 0.01 when f(z) is transcendental. It is not clear what value W should have if f(z) has both algebraic and transcendental terms. Igarashi gives only an abbreviated explanation of how this works. Presumably, the basic explanation is similar to the one proposed above for polynomials. Igarashi also gives only an abbreviated explanation for the choice of W. W=1.0 appears to be based on the assumption that the evaluations of algebraic functions and polynomials incur similar round-off errors, so the same stopping criterion can be used for both. But Igarashi also suggests that W=0.5 can be used instead to relax the stopping criterion to allow for errors in experimental data and/or the conversion of experimental data from decimal to floating-point binary. Finally, Igarashi chooses W=0.01 to further relax the stopping criterion for transcendental functions in order to account for the truncation errors incurred by the intrinsic transcendental functions used to evaluate A(zi), D(zi) and C(zi).Igarashi  providesf(zi), f′ (zi) and f′′(zi) are needed by the iteration procedure itself, and D(zi) and C(zi) are needed by the stopping criterion. Thus, Igarashi's stopping criterion adds two-thirds to the work load per iteration. The criterion is included in the comparisons reported in §7.If a third order iteration procedure, such as Ostrowski's method (e.g. Orchard ), is use3.3zi=xi+jyi is accepted as a root of the complex polynomialc0|<ϵg0(1+ϵ)n5 and |d0|<ϵh0(1+ϵ)n5. c0, d0, g0 and h0 derive from the recurrences:ϵ=2t− is the machine epsilon, i.e. t is the bit length of the floating-point significand, e.g. t=53 for IEEE double-precision. A rigorous derivation of this stopping criterion is given by Grant & Hitchins . The calculation is demonstrated below.sm is the length of the floating-point significand used, e.g. sm=53 bits and sm=113 bits, respectively, for IEEE double-precision and quad-precision. qm is the order of the iteration procedure to which the stopping criterion is applied, e.g. qm=3 for Laguerre's method and Ostrowski's method.Also in stopping criterion #1, a,b. These examples use iterates z1, z2 and z3 from sm=24 bits, as in IEEE single-precision. Also shown are the corresponding iteration increments e2=|z2−z1| and e3=|z3−z2|. A trailing ‘b’ designates a binary number.Stopping criterion #1 is explained with reference to table 2s2 in table 2a is the number of MLBs of z1 and z2 following iteration #2. s2 is equal to the number of leading zero bits of e2 (i.e. 6) plus a fraction produced by (i) removing the radix point and the leading zeros from e2 to produce the bit-string 101 1111 0001 1110 000b, (ii) taking two's complement of the bit-string to get 010 0000 1110 0001 111b, and (iii) placing a radix point in front of the complement to produce the binary fraction 0.010 0000 1110 0001 111b = 0.256893. This fraction is added to the number of leading zeros of e2 to get s2=6+0.256893=6.256893, as shown in table 2a. s2=6.256893 states that z1 and z2 have 6.256893 leading bits in common. The not function in table 2a is used as shorthand for the above procedure for finding the fractional part of s2. A simple numerical procedure for calculating s is outlined in Nikolajsen . The criteria are applied in the following order of descending efficiency: #3, #1, #2 and #4. The order of application affects the percentage usage of the criteria because two criteria are often satisfied simultaneously.ei>ei−1 is replaced by ei≥ei−1 because ei>ei−1 fails to activate when ei approaches zero monotonically and then remains equal to zero. For complex root finding, Ward's criterion is applied to the real and imaginary iteration streams separately because Ward was found in one instance to be triggered prematurely when applied directly to the complex iteration stream.Ward's stopping criterion zi+1is a root if zi+1=zi. Without this, G&H does not get invoked when the start value by chance hits a root.G&H's stopping criterion is supplemented by the following criterion: 7.2a shows the total number of determinant evaluations needed to find all the eigenvalues of dense, random, non-symmetrical matrices of orders 5 through 250 using JLN's and Ward's stopping criteria. The same random matrices are used for both. The matrices are reduced to upper Hessenberg form to allow the eigenvalues to be extracted by Laguerre's method, as outlined in Nikolajsen 2. All the roots are double roots and the single root Laguerre formula is used to slow the convergence rate to linear for half the roots in order force stopping #2 and #4 into action. The results are shown in In the previous polynomial examples, convergence is so rapid that stopping criteria #2 and #4 have hardly been used. Their use is demonstrated here by calculating the roots of polynomial type 2-type polynomials. The 19.2% usage of stopping criterion #4 is still surprisingly low. The raw data suggest the following explanation. The lower accuracy of double roots accelerates the loss of accuracy of the deflated polynomials. As a result, the double roots still to be extracted gradually lose accuracy and split apart to become single roots, whose convergence rate is much greater. As a result, the faster stopping criteria, #1, #2 and #3, gradually take over from #4. This is also reflected in figure 5a by the progressive levelling off of both the Ward and the JLN lines as the number of function evaluations per root drops due to the greater speed of convergence of the emerging single roots.The percentage usage of JLN stopping criteria #1, #2, #3 and #4 changes from 74.3%, 0.5%, 25.1% and 0%, respectively, for b and 4b shows that this remains at 82. The reason appears to be that, once the double roots have split, any further deterioration in their accuracy is greatly reduced because they have become single roots that can be extracted with twice the number of MLBs. Of course, the first roots extracted will still be double roots, so the maximum accuracy cannot exceed half the single root accuracy, as also indicated by comparing figures 5b and 4b.A corresponding reduction might be expected in the maximum polynomial degree at which all the roots can be matched unambiguously to their exact counterparts. But comparison of figures 5b again confirms that both are equally accurate. The exception is the blip in the Ward graph at polynomial degree 52. The raw data suggest that this is due to a small variation in the linear rate at which MLBs are gained when the first of a pair of double roots is extracted by the single root Laguerre formula. In this particular case, a very slow constant gain of 1.8 bits per iteration is interrupted by a premature loss of 0.6 bits, which triggers Ward's stopping criterion prematurely, reducing the number of correct decimal digits of the 21st root from 12.5 to 11.7 . As this small error propagates through the subsequent deflations, it gradually reduces the accuracy of the subsequent roots to the point where the number of correct decimal digits of the poorest root drops from 8.8 to 6.1, as indicated in figure 5b. This could be avoided by putting a lower limit on the loss needed to trigger Ward, but that would reintroduce the problem which was solved in §7.1 by replacing ei>ei−1 by ei≥ei−1.The coincidence of the JLN and Ward graphs in figure 5si+2≤si+1 by si+2≤si+1−1 in stopping criterion #4. But that would lead to triggering difficulties in many other cases. The conclusion is that neither JLN stopping criterion #4 nor Ward is fail-safe at extremely slow convergence rates that can occur when the single root Laguerre formula is applied to a double root. But this is unlikely to happen in practice, when a sustained linear rate of convergence would be used to trigger a switch to the double-root Laguerre formula.JLN stopping criterion #4 avoids this failure only by chance, i.e. because tiny differences between the iterates (caused by faster termination of previous roots) happen to produce a Laguerre iteration sequence that does not include a tiny premature loss of digits. This potential problem could be avoided by replacing p2(z). In other words, the advantage of JLN over Ward, in terms of efficiency, is reduced from 37.1% for p2(z)-type polynomials to 4.76/42.70=11.1% for [p2(z)]2-type polynomials. The main reason is the more frequent need for JLN stopping criterion #4, whose efficiency is no better than Ward's. The reduced advantage of JLN is also evident from the closer proximity between Ward and JLN in figure 5a compared with figure 4a.The total number of function evaluations is 39 288 with Ward and 34 905 with JLN. The total number of roots found is 8+12+16+⋯+84=920. Thus, the average number of function evaluations per root is 39 288/920=42.70 with Ward and 34 905/920=37.94 with JLN. Thus, JLN reduces the number of function evaluations per eigenvalue by an average of 4.76, compared with 7.32 for Polynomials with several other distributions of known roots were also tested, as were polynomials with random roots, for a total of approximately 739 polynomials of different degrees and levels of ill-conditioning with a total of approximately 60 227 roots. All the tests confirm the findings of this and the previous sections. Results are shown in the electronic supplementary material.7.4W=0.01 for transcendental functions and W=0.5 or W=1.0 for algebraic functions.)Igarashi's, Ward's and JLN's stopping criteria are implemented in a general nonlinear root finder based on Ostrowski's square root iteration formula in the complex domain (e.g. ). (‘Igarf1 is so simple that its exact roots are known. f2(z)=0 is one of two purely transcendental frequency equations for a beam with three equal-length spans on four pinned supports [f3(z)=0 is the frequency equation for lateral vibration of a beam with flexible, pinned supports at each end [Results are presented for the following sample functions:supports . f3(z)=0each end .W=0.01 for purely transcendental functions like f1 and f2 but does not make it clear which W-value should be used for functions like f3, which has both algebraic and transcendental terms. A preliminary value of W=0.01 is used for f3 because it is the least stringent and therefore the most likely to trigger Igarashi. This is an attempt to avoid Igarashi's failures to stop when a root has been found, as experienced earlier for both matrices and polynomials.Igarashi prescribes the use of f1, f2 and f3. The average number of function evaluations per root are shown in Two hundred and fifty roots are extracted for both f1, f2 and f3. Inspection of the raw data shows that this is because (1) neither Iga, Ward nor JLN terminate the iterations until convergence has been completed and (2) in all cases, the first converged iterate and all subsequent iterates are exactly equal. No attempt was made to explain (2): the workings of the root solvers are beyond the scope of this paper. The important conclusion here is that all three sets of stopping criteria provide equal accuracy. Igarashi's previous failures to stop are not encountered with functions f1, f2 and f3.In terms of accuracy, Iga, Ward and JLN provide the exact same values for all 250 roots of both p1(z) is the polynomial tested in §7.3.1. The subtraction of z5/2 is used as a simple means of making f4 non-polynomial without introducing discontinuities in the f4-derivatives needed by Ostrowski. f4 has n roots, one for each of the n intersections between p1(z) and z5/2.Purely algebraic, non-polynomial functions are not common in practice but, for completeness, the roots of the following such functions are found:n=8,12,16,…80, with W=1.0 used for Igarashi. f4 turns out to be quite a challenge for Ostrowski's method, which fails to find the nth root for n=28, 44, 60 and 68, regardless of which set of stopping criteria is used. The four peaks in figure 7a are due to the additional iterations carried out in the fruitless search for the final root. The three topmost data points of each peak are the peak values for Iga, Ward and JLN, in the order shown for n=68.The results are shown in b gives the relative accuracy of the stopping criteria in terms of log10|( f4)|max, i.e. the logarithm of the largest absolute f4-value at the roots. The exact f4-values at the roots are of course zero. Any log10|( f4)|max-value less than −34 is set to −34 .The exact roots are unknown, so figure 7b, the accuracy of all three sets of stopping criteria is virtually the same except for (i) the tiny dip in the Iga line at n=28, (ii) the huge dips in the Iga line at n=72 and 80, and (iii) the huge dip in the Ward line at n=80. These dips do not signal any momentary improvement in accuracy. Rather, they occur for the following reasons:n=28, 32 roots for n=72, and 35 roots for n=80,(1) when Igarashi is applied, Ostrowski is only able to find 21 roots for n=80, and(2) when Ward is applied, Ostrowski is only able to find 35 roots for n roots are found, log10|( f4)|max becomes unrepresentatively small. This is because the roots are found roughly in ascending order of modulus, and the size of each log10|( f4)|-value is roughly proportional to the modulus of the corresponding root, so when only the small-modulus roots are found, the large log10|( f4)|-values are missing.(3) when so few of the As shown in figure 7These problems appear to be related to relatively weak global convergence properties of Ostrowski's method. They are therefore beyond the scope of this paper and will not be pursued. It is also not clear why these problems are not encountered when JLN's stopping criteria are applied.n=28, 60, 64 and 72, respectively. With W=0.5 instead of 1.0, the total number of failures grows to 32. As before, the iterations are stopped automatically by the root finder, so the failures do not affect the apparent accuracy of Iga. But, as before, these failures are deemed to be unacceptable, thus disqualifying Igarashi's stopping criterion in its current form.Inspection of the raw data shows that Iga fails to stop, when a root has been found, on a total of 25 occasions: once, twice, three times, 10 times and 11 times for The average number of function evaluations per root is 12.391 with Ward and 9.070 with JLN for all the root extractions related to 8.(1) Grant & Hitchins tended to trigger prematurely, leading to multiple failures. Igarashi occasionally failed to trigger, leading to multiple redundant iterations and uncertainty as to whether a root had been found. Both were therefore rejected in their current formulations.(2) Ward and JLN were the only criteria that did not fail. Both provided the maximum possible accuracy of the results but JLN did so more efficiently, reducing the number of function evaluations by one-third without any deterioration in the accuracy of the results.(3) Ward's criterion is by far the simplest and most easily implemented. So when simplicity is more important than efficiency, Ward's criterion is preferable. JLN's criteria are preferable in all other cases.A new set of stopping criteria for iterative root finding and eigenvalue extraction has been presented, which terminates the iterations immediately when no further improvement of the results is possible. The new criteria, called JLN, have been tested numerically against the existing stopping criteria of Igarashi ,5, GrantThe range of testing reported here is of necessity limited and must be regarded as preliminary. But it is considered to be sufficiently convincing to warrant further consideration of the JLN stopping criteria when high efficiency is desired.A numerical implementation of the JLN stopping criteria is available from the author on request.


These heterostructures offers excellent current-carrying abilities whilst offering the prospect of a fast, low cost and easy methodology for device applications. The use of ND monolayers is also a compatible technology for the support of large area graphene films. The nature of the C-H bonds between graphene and H-terminated NDs strongly influences the electronic character of the heterostructure, creating effective charge redistribution within the system. Field effect transistors (FETs) have been fabricated based on this novel herterostructure to demonstrate device characteristics and the potential of this approach.Graphene on hydrogen terminated monolayer nanodiamond heterostructures provides a new way to improve carrier transport characteristics of the graphene, offering up to 60% improvement when compared with similar graphene on SiO The detailed material properties of graphene on ND surfaces with and without hydrogen termination treatments have been investigated using Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. It has been shown that the hydrogen termination treatment not only removed surface contamination from as-deposited monolayer NDs, but also provided a suitable linkage between ND and the graphene layer to form a conductive path, as is demonstrated by impedance spectroscopy (IS) measurements. Also, here the carrier mobility of graphene on hydrogen terminated ND (GrHND) has been compared to graphene on hydrogen terminated SCD (GrHSCD). In addition, to Hall mobility measurements, top-gate graphene transistors with two different gate lengths of 200 nm and 500 nm have been fabricated using a focused ion beam (FIB) tool; and thus, this study has demonstrated a new approach for commercial graphene transistor fabrication.In this paper, a cost effective and mass producible method to fabricate monolayer ND films that are capable of tuning the properties of graphene for the fabrication of Field-Effect Transistors (FETs) is demonstrated. Compared to similar graphene transferred onto SiO2/Si substrate (300 nm SiO2) is shown in 2/Si substrate with an ND-water solution, then an ultra-sonic bath treatment to attach the NDs, followed by drying. Thermal hydrogenation was then performed . The surface coverage and roughness of the ND layers were determined by atomic force microscopy (AFM), as shown in etc20−1 is characteristic of the C = O stretching band involved in carboxylic acid groups and anhydride functionalities−1 region is attributed to the OH of adsorbed water. Various peaks relating to other ND surface groups  are observed in the 1000–1500 cm−1 range−1 have been reduced. The free and adsorbed -OH peaks have disappeared in the region of 3000–3600 cm−1, while the peak at 2923 cm−1 has become much stronger, being assigned to the C-H stretching of the hydrogenated ND surface coupled with the CHX bands at 1461 cm−1 (CH2) and 1377 cm−1 (CH3)A schematic view of the GrHND heterostructures supported on a SiOubstrate 0 nm SiO22g phonon at the Brillouin zone centre and the D peak is due to the breathing modes of an sp2 phase and defects for those activations3/sp2 bonded phase can be expected for graphene on NDs and H-NDs. The dangling bonds from ND and H-ND surfaces evidenced in the FTIR results lead to the observed shifts in the Raman peaks as compared to unsupported graphene layers392, here, the G peak positions for the graphene-ND heterostructures have an upshift from 1581 cm−1 to 1586.2 cm−1 and 1585.7 cm−1 for untreated ND and H-ND substrates respectively41−1 for ND (2698 cm−1) and H-ND (2690 cm−1) substrates, respectively, due to hole doping−1 2D peak of graphene on SiO2. The 2D peak widths for both heterostructures are within 25–28 cm−1 range, suggesting the graphene is monolayer in nature2D/IG can be used to provide additional information2D/IG2D/IG for both heterostructures are 1.06 and 1.17 respectively, proving electron transfer between the interface and thus high levels of doping of the graphene has occurred; for low doping the 2D peak is expected to be 3–5 times stronger than the G peak3/sp2 ratio3 signal, leading to the heterostructure here being formed from sp3/sp2 carbon mixtures. Both FTIR and Raman results show a clear modification occurs to the graphene lattice with H-termination treatment of the NDs. The charge transfer between graphene and NDs introduces disorder and high doping to graphene, hence tuning the graphene electronic properties.Raman spectroscopy was next used to study the graphene-ND hetrostructure to investigate the chemical nature of the carbon materials present332 and sp3 atoms present2 hybridized C atoms in ND (graphite structure) and graphene443 hybridized C atoms due to the formation of C-H and the C–C bonds in the ND film4446472 contribution since NDs are partially covered by a thin layer of graphite3 hybridization of the deposited carbon by saturation with hydrogen atoms, whilst thermal annealing  leads to graphitization and an increase of the sp2 phase463 phase contributing to the D peak143 bonds are likely to affect the translational symmetry of C = C sp2 bonds in graphene, which is similar to the effect of hydrogenating graphene3 coverage was determined as θ/(1 + θ) where θ is defined as the intensity area of sp3 and sp2 peaks3 coverage was found to be ~19%, while for graphene transferred on H-ND films the sp3 coverage increased to ~37%. As a result, the hydrogen terminated ND film has also indirectly hydrogenated the monolayer graphene to an H-coverage of approximately 37%−19% = 18%. , where A0 is the pre-exponential factor; KB is the Boltzmann constant and Ea is the activation energy representing the energy barrier for the thermal activation process55The electrical characteristics of the heterostructures were examined using AC impedance spectroscopy (IS) measurements. Two Au/Ti (300 nm/10 nm) contacts were deposited on the top-side corners of square samples. IS was carried out in vacuum at temperatures ranging from room temperature to 500 C as shown in 5150532/Si is 132 cm2/Vs  segments, and amorphous carbon at the grain boundaries13 cm−2 to a highest point of 1.3 × 1013 cm−2 at 400 K, then dropped slightly to 1.2 × 1013 cm−2 at 450 K. The carrier density of GrHSCD shows a linear increase with temperature from 6.4 × 1012 to 8.5 × 1012 cm−2 at room temperature to 450 K.The sheet resistance and carrier densities for both heterostructures have been plotted against temperature as shown in 2O and CO2 to produce H3O+, HCO3− and H+ layers shown in Although GrHSCD has lower a carrier density which enables an increase in the mobility2O3 dielectric layer with 10 nm thickness was grown by atomic layer deposition (ALD Ultratech Fuji F200). Detailed fabrication information is given in the fabrication method section. et al. measured the Dirac point voltage of their graphene FETs formed on DLCet al. suggested that the aluminium nanoparticles present in their work tuned the Dirac point, which then induced n-type dopingds/dVg) × (L/WCiVds), where L is the channel length (5 μm), W is the channel width (500 nm) and Ci is the areal capacitance per unit area between the channel and gate (8 × 107 F/cm2)2/Vs, which is similar to the measured Hall mobility.Top gate FET devices were fabricated on GrHND heterostructures with different gate channel sizes of 200 nm and 500 nm, respectively. A focussed-ion-beam tool, giving a potential feature size smaller than 10 nm, was used to define the source, drain contacts, gate electrodes. FIB was also used for local material deposition for contact formation using tungsten carbide (WC). A top-gate Al3/sp2 carbon present has changed. From IS measurements performed at various temperatures, it is indicated that the hydrogen links both ND and the graphene layer to create a charge transfer process which induces a conductive interface layer between graphene and ND. The mobility of GrHND increases some 60% compared with similar graphene on SiO2/Si and is comparable with observations GrHSCD, which is not viable for commercial applications. Moreover, over the whole temperature range, GrHND demonstrated stable carrier mobility compared to GrHSCD which is vital for commercial applications. Whilst the mobility of the CVD-derived graphene layers here was modest, it can be expected that the advantage of the use of a supported ND layer, over SCD, DLC or SiO2/Si would be maintained for higher quality CVD-grown graphene layers as they emerge as commercial materials. FETs fabricated on this novel type of herterostructure GrHND demostrated comparable characteristics to those FETs on graphene-SCD, whilst offering the prospect of low cost large area production of graphene electronics.A systematic study of the properties of graphene-ND heterostructures has been carried out and the operation of an FET on GrHND structures demonstrated. Due to the prospects for low cost, easy deposition and an associated high thermal conductivity, monolayer ND is a promising candidate as a support material for graphene. It appears a better solution than other novel materials such as SCD or UNCD in case of the need for high current-carrying device capacity and mass reproducibility. The properties of graphene on NDs with and without H-termination have been investigated using FTIR, Raman spectroscopy, and XPS. The C-H bond present on H-terminated NDs, strongly influences the heterostructure properties that result from depositing graphene on the NDs. The presence of H-termination leads to the appearance of a so-called D peak, and to a shift in the G and 2D peaks, suggesting the ratio of sp2/Si substrates with 300 nm thickness of SiO2. Nanodiamonds were supplied prepared in solution from ultra-dispersed detonation nanodiamonds . Deposition involved immersing the substrate material in the ND solution (0.05 g/L of NDs) followed by sonication (10 min). Hydrogen termination for NDs was performed using a Seki AsteX AX6550 reactor with the following parameters for 5 hours: microwave power, 600 W; H2 200 sccm; pressure 10 Torr; temperature 500 C. CVD grown monolayer graphene on a copper foil (Graphene Supermarket) was coated with PMMA. The PMMA-coated graphene was treated with Iron nitrate solution to etch away Cu foil. The floating PMMA coated graphene was removed and cleaned in DI water to transfer onto H-terminated NDs samples. The PMMA was removed by acetone.As shown in The surface morphology of the various films measured over 10 μm × 10 μm and 1 μm × 1 μm regions was measured using scanning atomic force microscopy  in tapping mode for the H-terminated NDs. The chemical composition and bonding states of the films were characterized using Fourier transfer infrared spectrometry  in transmission mode and X-ray photoelectron spectroscopy  with the Al K α line as the exciting source. A micro Raman spectroscopy system (Renishaw Invia) operating at 514.5 nm was used to study the film microstructure, where the laser output power used was 1 mW. Impedance spectroscopy was applied in the range of 0.1 Hz to 10 MHz in a vacuum at elevated temperature from room temperature to 500 C using an Autolab electrochemical system. Hall Effect measurements  were performed to determine graphene carrier mobilities using the “van der Pauw” method, where by four Ohmic metal contacts (10 nm Ti-300 nm Au) were placed on the topside of the graphene layers at room temperature.2) and material-specific preferential milling can be performed by introducing reactive gases in the vicinity of the electron or ion probe. The electron column delivers the imaging abilities of the SEM which is less destructive than FIB imaging. SEM imaging of the graphene sample before milling identified clean area suitable for FET fabrication. Keep one clean graphene area 5 μm × 10 μm then other surrounding area around 100 μm × 110 μm were milled by using a 30 kV Ga+ beam with a current of 2 nA to remove the graphene and nanodiamond layers for FET definition. Before depositing S/D/G contact patterns, an insulator material (SiO2) was deposited on graphene surrounding area (50 nm thickness). Three contacts patterns 40 μm × 40 μm were coated by Tungsten Carbides with thickness of 200 nm using a 30 kV/2 nA beam. A final gentle “polish” with Ga+ ions (30 kV/ 5 pA) was used to clean the graphene area edge and remove side damage. Finally connect contact pattern with graphene area edge by WC using a 30 kV/50 pA beam. After the deposition of 10 nm Al2O3 layer by Atomic layer deposition (ALD), the Al2O3 layer on big gate pattern was removed then one narrow gate channel was built on top. In this study, the gate sizes are 500 nm × 5 μm and 200 nm × 5 μm.Graphene field-effect transistors were fabricated using a Carl Zeiss Focused Ion Beam Microscope and an Atomic Layer Deposition system. A dual-beam instrument (Carl Zeiss XB1540 cross-beam Focussed-Ion-beam microscope) was used to pattern samples without using masks, giving a feature size as small as 10 nm. The instrument combines a FIB and a SEM column in the same chamber and is fitted with a gas-injection system to allow local material depositions .


In this article, we have theoretically investigated the performance of graphene-hexagonal Boron Nitride (hBN) multilayer structure  to demonstrate all angle negative refraction along with superior transmission. hBN, one of the latest natural hyperbolic materials, can be a very strong contender to form a hyper crystal with graphene due to its excellence as a graphene-compatible substrate. Although bare hBN can exhibit negative refraction, the transmission is generally low due to its high reflectivity. Whereas due to graphene’s 2D nature and metallic characteristics in the frequency range where hBN behaves as a type-I hyperbolic material, we have found graphene-hBN hyper-crystals to exhibit all angle negative refraction with superior transmission. Interestingly, superior transmission from the whole structure can be fully controlled by the tunability of graphene without hampering the negative refraction originated mainly from hBN. We have also presented an effective medium description of the hyper crystal in the low-k limit and validated the proposed theory analytically and with full wave simulations. Along with the current extensive research on hybridization of graphene plasmon polaritons with (hyperbolic) hBN phonon polaritons, this work might have some substantial impact on this field of research and can be very useful in applications such as hyper-lensing. Negative refraction2346623434111314151314156Graphene, widely used in both photonics and plasmonics in a broad frequency range because of its unique and tunable optical properties, has been recently used in constructing HMMs10171820212223242526In this article, we have considered the graphene-hBN multilayer structure for constructing a hyper crystal. Graphene shows both better mobility and high relaxation time, if hBN is the substrate material20211314152830σ of graphene depends on frequency (ω), chemical potential (μc), relaxation time (τ) and temperature (T). It can be determined using the Kubo formalisms34The optical conductivity The tangential permittivity of graphene is expressed as,ε0 is the free space permittivity and tg represents the thickness of monolayer graphene. As graphene is a two dimensional material, the normal electric field cannot excite any current in the graphene sheet. So the normal component of the permittivity is given by εg,⊥ = 137εg,⊥ can be approximated as the permittivity (~2) of graphite. In this article, the graphene that we have considered has only a few layers . The relaxation time of graphene indicates its purity, and it can vary from fs (femto-second) to ps (pico-second) range. We have considered the relaxation time as τ = 200 fs in all calculations. This is a reasonable value as considered in literature for different graphene based devices1017181516Here ε∞ and γ indicate the high-frequency dielectric permittivity and the damping constant, respectively. These data have been taken fromεuu becomes negative.where u =  represents the transversal  and u = z represents longitudinal  axes. LO and TO respectively stand for longitudinal and transversal optical phonon frequencies. Hyper-crystals can be constructed by a stack of17−1. In hBN, two Reststrahlen bands are non-overlapping1315LO = 825 cm−1, ωTO = 760 cm−1) εxx = εyy > 0 and εzz < 0 and in the upper Reststrahlen band (URB),  εxx = εyy < 0 and εzz > 0 and thus leading to the Type I and II hyperbolic response, respectively.For a general one dimensional anisotropic crystal or hyper-crystal, each layer has the permittivity tensor of the form,The basic Bloch equation for a p-polarized wave in case of an anisotropic photonic crystal has been derived as,, can be given by,Here dk0stands for the wave-number in free-space.where, zd ≪ 1 is the sufficient condition for effective medium theory. But for hyper-crystal, the situation is slightly differentk waves (k > k0). So, effective medium theory cannot properly describe the physical properties in hyper crystal, such as the band gap, originating from the high-k waves in either or both layers. This failure of effective medium theory in hyper crystal0) and for moderate high-k waves (up to k ~ 20 k0), effective medium theory should still work, where layer thicknesses (d1 and d2) and wavelength (λ) play a critical role. Effective permittivity tensor for a hyper-crystal can thus be derived from Now, for isotropic case, where both layers have isotropic permittivity , k occurs only in the lower Reststrahlen band (LRB) where hBN shows type I hyperbolic behavior. In the upper Reststrahlen band (URB), hBN shows type II hyperbolic behavior and propagating waves  (k < K0) are not allowed to propagate in either hBN or GhHC. The perpendicular permittivity of graphene is constant because it does not depend on the chemical potential of graphene. As a result, the effective perpendicular permittivity of GhHC is independent of the chemical potential of graphene. On the other hand, the effective tangential permittivity of GhHC is a strong function of the chemical potential of graphene. With higher chemical potential, the effective tangential permittivity of GhHC reduces from bare hBN to a value closer to the permittivity of ambient medium (air). TO = 760 cm−1). But the lower value of the imaginary part of the perpendicular permittivity at the upper edge of the LRB (ωLO = 825 cm−1) plays an important role in absorption as discussed later on.z,hc) can be given by,From To demonstrate whether effective medium theory works, and to what extent if it does, here we do not put any arbitrary tensor values in ωc = 1600 cm−1 and ωc = 800 cm−1 respectively for graphene’s chemical potential, μc = 0.5 eV. Other values of chemical potential of graphene can also be chosen for GhHC. Here, we have chosen μc = 0.5 eV arbitrarily for illustration purpose. In ωc = 1600 cm−1 is also shown. The shaded regions indicate the regions where no propagating wave is allowed. The red symbols represent the exact calculation (ωc = 800 cm−1 (in the LRB) and ωc = 1600 cm−1 (in the URB), hBN behaves as a type-I and type-II hyperbolic material, respectively. From 170).culation  and the culation . At ωc =Any (meta) material which possess hyperbolic dispersion , negative refraction especially the all-angle negative refraction will be available from such (meta) materials23789783rs,hc) and wave-vector  as a function of incidence angle at wavenumber 810 cm−1 for different values of chemical potential of graphene. rs,hc) as a function of wavenumber and incidence angle for μc = 0.25 eV. The black dashed lines indicate the angle of refraction equal to zero (Transition between positive and negative refraction). Only in the LRB (760 cm−1 ≤ ω ≤ 825 cm−1), the effective perpendicular permittivity of GhHC becomes negative .4445ene. cf. . But in ene. cf. . But in ene. cf. , effectiene. cf. . As a reene. cf. ; εxeff bAlong with transmission, we want to clearly mention about the absorption properties of GhHC. Although the resonance of absorption of both the hBN and GhHC is at the beginning of the lower Reststrahlen band (LRB) cf. , the max994445y) of a p-polarized wave, incident from air to hBN and GhHC  using EMT. z,hc)/Im is a well-known and accepted value for characterizing materials which possess negative refraction9TM,hc) for TM polarization and the figure of merit (FOM) respectively calculated using the effective medium approximation as a function of wavenumber for different potential values of chemical of graphene. Although for higher chemical potential values, the FOM decreases. But higher transmission is achieved for higher chemical potential values of graphene which mainly occurs due to the reduction of reflection at the ambient medium (air)-GhHC interface.The FOM = Re. We have found that an all-angle negative refraction is achievable in such a hyper crystal with much higher transmission compared to bare hBN. Transmission and negative refraction properties can be controlled by tuning the chemical potential of graphene. Here negative refraction originates from the optical properties of hBN and transmission from the structure can be fully controlled by graphene without hampering negative refraction. Such properties are quite remarkable, and experimental demonstration of such phenomena should be possible with current experimental setup. We have also presented an effective medium description of the hyper crystal in the low-k limit, which might be useful in theoretical calculations and it has been validated by numerical calculations. We believe that, with the current ongoing extensive investigation on the hybridization of graphene plasmon polaritons with (hyperbolic) hBN phonon polaritons2223242526How to cite this article: Sayem, A. A. et al. Negative Refraction with Superior Transmission in Graphene-Hexagonal Boron Nitride (hBN) Multilayer Hyper Crystal. Sci. Rep. 6, 25442; doi: 10.1038/srep25442 (2016).


The development of chemical strategies to render graphene viable for incorporation into devices is a great challenge. A promising approach is the production of stable graphene dispersions from the exfoliation of graphite in water and organic solvents. The challenges involve the production of a large quantity of graphene sheets with tailored distribution in thickness, size, and shape. In this review, we present some of the recent efforts towards the controlled production of graphene in dispersions. We also describe some of the chemical protocols that have provided insight into the vast organic chemistry of the single atomic plane of graphite. Controlled chemical reactions applied to graphene are expected to significantly improve the design of hierarchical, functional platforms, driving the inclusion of graphene into advanced functional materials forward. Various methodologies for the production of graphene and chemically modified graphene have been described during the last years . Among t2 lattice is partially disrupted into layers containing sp2–sp3 carbon atoms. The most drastic example is probably graphene oxide (GO), where the extreme oxidation of the carbon lattice causes a detriment in the electrical and mechanical properties of the sheets [To exfoliate graphite by wet chemistry techniques, it is necessary to decrease the π–π staking interactions between the graphene layers. In achieving this, the spe sheets . As consTo date, the exfoliation of graphite in dispersions has been obtained by a number of mechanochemical methods. Each one of these strategies presents advantages and disadvantages depending on the type of application. In this contribution, we summarize some of the chemical procedures used to obtain graphene dispersions for various applications. Special emphasis is placed on the liquid-phase exfoliation of graphite by ultrasonic techniques, since this is a straightforward process for the production of clean graphene layers ready for organic functionalization. Importantly, the exfoliation of graphite by ultrasonication is a very versatile technique that has already demonstrated its effectiveness in dispersing graphene in solvents with different physical and chemical properties. This flexibility allows the incorporation of additives such as surfactants , antioxiAs previously mentioned, a number of chemical strategies can be followed for the production of graphene in dispersions. Until now, the most widely used methodology has involved the production of GO . In thisAs an alternative, charged species can be used to induce the exfoliation of graphite. For example, the addition of electrons from alkali metal intercalation compounds resulted in the spontaneous exfoliation of graphite in water or polar solvents. This procedure yields stable solutions of negatively charged graphene sheets .As charged, intercalating species, ionic liquids are considered a green alternative. The negligible vapour pressure, thermal stability, wide electrochemical potential window, good conductivity, recyclability, and the high dielectric constant of ionic liquids induce the exfoliation of graphite by weakening the π–π stacking interactions. Indeed, the use of ionic liquids is considered a highly versatile and industrially scalable method for the preparation of graphene nanomaterials .Some neutral molecules are also able to exfoliate graphite by producing polarized π-systems . For exaThe methodologies used for the exfoliation of graphite described thus far might pose a challenge to the further modification of graphene layers by covalent organic reactions. The presence of additional chemical species could yield unexpected products. Thus, different approaches are followed for the production of clean graphene sheets. For example, graphene can be produced by supercritical solvent exfoliation of graphite. In this procedure, solvents reach or exceed their critical point, presenting outstanding wetting properties, low interfacial tension, low viscosity, and high diffusion coefficients. Under these conditions, graphite is exfoliated as high-quality graphene .A versatile and simple strategy to exfoliate graphite in liquid phase is based on the use of ultrasonic wave treatment. Ultrasound techniques have important applications in a wide range of materials synthesis strategies . The phy−2). This relatively high surface energy results in solvents with high boiling points and high surface tensions, for example, N,N-dimethylformamide (DMF) and N-methyl-1,2-pyrrolidone (NMP), which are well-known as good dispersing solvents for carbon nanostructures such as fullerenes and carbon nanotubes (CNTs) [Ultrasonic processes are successful at exfoliating graphite only if the net energetic cost of the process is very small. The enthalpy of mixing depends on the affinity between graphene layers and solvent molecules. Then, in order to obtain high yields of exfoliated graphite, the surface energy of the solvent must compete with the surface energy of graphite (≈70–80 mJ∙ms (CNTs) –25. UsinAs an alternative, it is possible to prepare graphene dispersions in chloroform, acetone, or isopropanol. These are all volatile solvents that have generally lower surface tensions, and are therefore more easily evaporated. Unfortunately, in order to achieve graphene layers of reasonable concentrations in volatile solvents, it is necessary to increase the ultrasonication time, which increases the oxidative processes that cut the graphene sheets into smaller layers, resulting in a higher content of oxidized carbon atoms .The exfoliation of graphite in water is also possible by adding surfactants as stabilizing agents. For example, graphite and sodium cholate were ultrasonicated in water for long periods up to 400 h . This prN-(2-mercaptopropionyl)glycine (tiopronin) was reported [As previously mentioned, ultrasonication is a highly energetic process that produces strongly oxidizing chemical species, such as radicals and peroxides. These radicals typically oxidize carbon atoms in graphene close to the defects and at the edges. Under prolonged periods of ultrasonication, the oxidation can cut graphene sheets into small pieces. A well-known strategy to mitigate the damage induced by radicals in different biological processes is through the addition of antioxidant. Following this approach, the ultrasonication of graphite upon addition of reported . TiopronAn important potential application of exfoliated graphene is the tailored production of other carbon nanostructures such as fullerenes  and CNTsThe proposed mechanism for the formation of the MWNTs from graphene is as follows : (1) theFrom an organic chemistry perspective, graphene is a very exciting material as it can be functionalized on the two faces, at the edges and at the defects. Initially, graphene was expected to behave as a chemically inert surface, similar to graphite. However, it was experimentally and theoretically shown  that funThere are two types or graphene edges: zigzag and armchair. Zigzag edges are considered more reactive as compared to the armchair ones . DefectsThus, reactions occur at both faces of the graphene layer and different types of edges show different chemical reactivity. Additionally, defects such as vacancies, adatoms or substitutional atoms might act as active chemical sites on the graphene layer . These fThe covalent organic functionalization of graphene is necessary for several purposes. For example: (1) to increase the dispersibility in common organic solvents and water and (2) to combine the properties of graphene with the properties of other functional materials such as nanoparticles, polymers or chromophores –39. The Organic reactions might present different reactivity on the graphene surface when compared with other carbon nanostructures. The characterization of functionalized graphene layers typically requires the use of advanced analytic techniques. Graphene layers in dispersions are normally present at low concentrations and functional groups appear only sporadically. Thus, high-resolution techniques such as X-ray photoelectron spectroscopy (HR-XPS) and HR-TEM are very useful for the identification of functional groups.2 lattice of carbon nanostructures is the azomethine ylide [A common organic reaction used for the functionalization of carbon nanostructures such as fullerenes, nanotubes, nano onions, nano horns and currently graphene, is the use of dienophiles as reactive species. One of the most successfully applied dienophiles to form covalent bonds with the hexagonal spne ylide . This inThe 1,3-dipolar cycloaddition, performed on graphene by employing an aldehyde and an α-amino acid as precursors, attacks both graphene faces and edges. This reactivity was observed by introducing protonated terminal amino groups that selectively bind gold nanorods allowing the recognition of the reactive sites by low resolution TEM . The HR-In order to acquire more knowledge related to the organic chemistry of exfoliated graphene by ultrasonication, carboxylic groups where introduced on the graphene sheets by applying the 1,3-dipolar cycloaddition. This product was compared with fresh exfoliated graphene by the derivatization of both products with PAMAN dendrons using an amide-bond condensation reaction . Using AAs expected, for the 1,3-dipolar cycloaddition reaction, the TEM micrographs showed Au NPs completely dispersed on graphene faces and edges. In contrast, for the amide-bond condensation reaction, Au NPs were mainly found on the graphene edges. These results were corroborated by HR-XPS analysis of the products. A higher concentration of functional groups was found in graphene functionalized by using the 1,3-dipolar cycloaddition. With this, we demonstrated that graphene sheets could be selectively functionalized on the borders or in the entire lattice .These results corroborate the success of the production of graphene layers by ultrasonication techniques. Oxidized carbon atoms on graphene sheets are introduced mainly on the edges of the layers when compared with GO. Additionally, the presence of the adatoms is not revealed by HR-XPS or HR-TEM.4POM) anionic catalyst by electrostatic interactions, as illustrated in As described above, organic chemical reactions can be chosen to attack the complete graphene sheets or mostly the edges. Strategies such as this enable the integration of the outstanding properties of graphene in different applications. For example, graphene–polyoxometalate hybrids are considered important materials for the development of artificial photosynthesis. The properties of graphene enhance the electrocatalytic performance of the catalytic polyoxometalate and minimize the applied overpotential, increasing the long-term robustness of the device . Recentl2 surfaces [N,N,N-trimethylbenzenammonium, as illustrated in 4POM in the charged functional moieties by complementary electrostatic interactions were introduced. Comparing the insertion of a shorter moiety by the diazonium arylation reaction with the positively charged dendrons attached by the 1,3-dipolar cycloaddition described before, we confirmed the interplay of electrostatic forces and π-electron clouds on graphene. The proximity of the Ru4POM to the graphene surface in the arylation reaction leads to a tight immobilization.In order to introduce higher loadings of functional materials, stronger chemical reactions for graphene functionalization can be achieved by the attack of radical species. As reactive radical species, aryl diazonium salts are attractive and popular sources. These radicals covalently graft to carbon spsurfaces . Using t4POM molecules were identified on functionalized graphene surfaces by low voltage aberration-corrected TEM (AC-TEM) [4POM anions on functionalized graphene was performed. When comparing the Ru4POM anions accompanying short carbon moieties with those attached to dendrons with longer carbon chains, a higher degree of motion and frequency were observed for the latter, as shown in Finally, the optical properties of graphene for the high contrast imaging of the two previously described graphene–polyoxometalate nanohybrids can be exploited. For this, Ru(AC-TEM) . FollowiSubstantial efforts have been performed for the mass scale-up and low-cost production of graphene. Thus far, wet chemical strategies have been demonstrated as the most efficient for the inclusion of graphene in low performance applications. To improve the physical properties of chemically produced graphene, it is necessary to gain better control of the experimental techniques by increasing the knowledge of the physical and chemical properties of both graphite and graphene. Likewise, it is critical to acquire detailed comprehension of the organic chemistry of the two dimensional material. The tailored chemical design of graphene platforms is essential for the development of new exciting functional materials.


Persistent infection of high-risk human papillomaviruses 16 (HPV16) has been considered as the leading cause of cervical cancer. In this study we assessed HPV16 sequence variation and genetic diversity of HPV16 variants in cervical cancer in Uigur women in Xinjiang, China. We analyzed the nucleotide sequences of the open reading frames of E6 and E7, and part of the open reading frames of L1 of HPV16 in Uigur women.Biopsies of histologically confirmed HPV16 infections with cervical cancer were obtained from 43 Uigur women in Xinjiang, China. E6, E7 and L1 genes of HPV16 of all samples were amplified and sequenced; the sequences were used in phylogenetic analysis of HPV16 variants.Our analysis revealed nine nucleotide changes in E6 (five changes), E7 (one change) and L1 (three changes) gene. The most frequently observed variations were T350G (79.1 %). One variation T295G (D64E) at E6 were detected in 6 cases . Deletion (464Asp) along with insertion (448Ser) were observed in L1 (100 %). Most variants were European lineage (97.7 %); only one belongs to Asia variants with common T178G (D25E) in E6 and A647G (N29S) in E7.The most prevalent HPV16 variants in the Uigur women we studied were of the European lineage. Our results indicate that HPV16 European lineage may serve as a harmful factor associated with the development and progression of cervical cancer.The online version of this article (doi:10.1186/s13027-016-0089-2) contains supplementary material, which is available to authorized users. Cervical cancer was the third most common cancer among women in the world, with 527,624 new cases and 265,653 deaths in 2012 [Persistent infection by high-risk HPV16, has been recognized as a critical etiological factor for cervical cancer , which iMultiple factors are involved in the development and progression of cervical cancer and most HPV16 infection can be removed by the immune system but a small proportion can progress to cervical cancer. Previous studies demonstrated that HPV16 variants increased the risk for progression to cervical cancer –10, but Based on the genomic analysis of HPV16, seven major lineages of HPV16 variants have been detected and are related to geographic areas: European (E), Asian (As), Asian American (AA), African 1 (Af1), African 2 (Af2), North American (NA)  and a reSeveral mutations in E6 and E7, L1 genes may have great influence on the efficiency of infection, viral antigenicity and immunogenicity. A number of studies have suggested that the non-European variants have an increased risk for progression to high-grade squamous intraepithelial lesions (HSIL) when compared with European variants , 10, 17.In Xinjiang, previous research showed that HPV16 was the most prevalent HPV type in Uigur women . We analBiopsies of histologically confirmed HPV16 infections with cervical cancer were obtained from 43 Uigur women, who attended the People Hospital of Kashi (southern Xinjiang) and the People Hospital of Autonomous region (northern Xinjiang) during the years 2011 to 2014. 20 of the 43 women were residents of the southern Xinjiang, and the other 23 were in the northern Xinjiang. All of the cases were identified as squamous carcinomas. The diagnosis of histopathological grades was examined independently by two gynecologic pathologists. Tissues were stored at 4 °C no more than 24 h after surgical removal and subsequently cut into small fragments and stored in liquid nitrogen for genomic DNA extraction. Informed consent was obtained from all patients and the study protocol was reviewed and approved by the ethics committees of the hospitals.DNA was extracted from the 43 cervical samples with the SK1252 Genomic DNA Isolation kit  according to the manufacturer’s instruction. HPV16 DNA was identified by polymerase chain reaction (PCR) using HPV16-specific primers  and 5 μl template DNA. The cycling conditions were: 94 °C for 5 min; 30 cycles of 55 °C for 45 s, 72 °C for 60 s, 94 °C for 15 s, 55 °C for 45 s, 72 °C for 5 min. Sequencing primers were listed in Table Fragments of HPV16 E6, E7 and L1 were amplified by PCR (Table PCR products were purified using SAP (Promega) and Exo I (Epicentre) and sequenced directly using ABI BigDye Terminator v3.1 Cycle Sequencing Kit on a DNA analyzer (ABI3130XL) at the Genesky Biotechnologies Inc . Single Nucleotide Polymorphisms (SNP) were analyzed by software Polyphred and were aligned with the prototype (GenBank: NC_001526.2)  and otheThe sequences of E6, E7 and L1 genes were subsequently assembled together using Sequence Matrixv 1.7.8 (http://dx.doi.org/10.1111/j.1096-0031.2010.00329.x). Phylogenetic trees were built by MEGA 6  The sequences generated in this study were deposited in GenBank with accession numbers KT959524 to KT959566 for E6, KT966608 to KT966650 for E7 and KT966651 to KT966689 for L1 genes.Forty-three full-length E6, E7 and 39 partial L1 genes (nt 6659–7023) were successfully amplified and sequenced. Sequences obtained were compared to the HPV16 prototype reference . Gene sequence variation in E6, E7 and partial L1 genes of HPV16 were shown in Table Six nucleotide changes were observed in E6 and E7 Table , which cOur sequence analysis showed that three base pair (ATC/GTC) were inserted into nt 6901–6902 along with three base pair (GAT) deleted at nt 6951–6953 in all samples (100 %); thus, a serine was inserted in amino acid (aa) position 449 whereas an asparagine was deleted from aa position 464. The sequence insertions and deletions of HPV16 L1 gene have not been reported before. The variant with the nucleotide insertion (GTC) at nt 6901–6902 was only observed in one sample. In addition, a silent change at nt 6989 (A to G) was observed in one sample  , and 1 case of Asia variant. HPV16 E6 L83V variant is prevalent in high-grade lesions and is associated with progression of cervical malignancy in Moroccan ; this vaIn good agreement with previous reports , 33, 34,Surprisingly, at the L1 gene, three nucleotide variations were found. One silent and two novel nucleotide variations are found; three base pair (ATC/GTC) were inserted at nt 6901–6902 along with 3 base pair (GAT) deleted at nt 6951–6953 in all cases (Table We investigated the genetic variation of HPV16 in Uigur women in Xinjiang. Our results show that HPV16 variant of European lineage is the most common type in Uigur women in Xinjiang, which is markedly different from elsewhere in China. Future studies should expand into larger population of patients to evaluate the association between HPV16 variants and the risk for cervical cancer, and to understand the evolution of HPV16 variants in Uigur women in Xinjiang.AA, Asian American; Af1, African 1; Af2, African 2; As, Asian; D, aspartic acid; DNA, deoxyribonucleic acid; E, European; E, glutamic acid; HPV, human papillomaviruses; HPV16, human papillomaviruses 16; HSIL, high-grade squamous intraepithelial lesions; Java, Javanese variant; L, leucine; N, asparagine; NA, North American; NE, Non-European; PCR, polymerase chain reaction; S, serine; V, valine; VLPs, virus-like particles


In this research, an improved psychrometer is developed to solve practical issues arising in the relative humidity measurement of challenging drying environments for meat manufacturing in agricultural and agri-food industries. The design in this research focused on the structure of the improved psychrometer, signal conversion, and calculation methods. The experimental results showed the effect of varying psychrometer structure on relative humidity measurement accuracy. An industrial application to dry-cured meat products demonstrated the effective performance of the improved psychrometer being used as a relative humidity measurement sensor in meat-drying rooms. In a drying environment for meat manufacturing, the achieved measurement accuracy for relative humidity using the improved psychrometer was ±0.6%. The system test results showed that the improved psychrometer can provide reliable and long-term stable relative humidity measurements with high accuracy in the drying system of meat products. Relative humidity is at all temperature and pressures defined as the ratio of the water vapour pressure to the saturation water vapour pressure (over water) at the gas temperature . There aThe psychrometric method of relative humidity measurement has been known for a very long time. The psychrometer measures the relative humidity based on evaporative cooling. The common structure of an aspirated psychrometer is shown in A prominent advantage of a psychrometer is its adaptability to measurement environments. Compared to modern electronic relative humidity sensors such as capacitive relative humidity sensors or resistive relative humidity sensors, the psychrometer is one of the relative humidity measurement devices that can be used in a dirty environment, can withstand state changes of water, and can provide a large range of measurement values. The psychrometer is used in different measurement environment in many current applications ,11,12,13However, as a relative humidity sensor in application system, the weakness of the psychrometer is its low measurement accuracy compared to electronic sensors ,15,16. TThe wet bulb temperature should be not a thermodynamic property, but, under certain conditions it well approximates the thermodynamic wet bulb temperature . The senet al. calculated the correlation coefficients of psychrometer with varying wind speeds. An aspirated psychrometer was designed and tested for broiler chicken houses [A number of prior works studied the psychrometer measurement method in different environments and the effect of different factors influencing measurement accuracy. Yin n houses . The tesn houses . Duuren n houses .et al. designed an intelligent dry and wet bulb relative humidity sensor, with simulation and test results demonstrating the accuracy to within 2% RH [Several researchers discuss the measurement accuracies of psychrometer. Ustymczuk and Giner examined the errors of psychrometric measurement of relative humidity and used an error propagation formula to convert temperature errors to relative humidity errors . Mittal in 2% RH . The accin 2% RH . The stain 2% RH . The tesin 2% RH . Nicholsin 2% RH . Nakahamin 2% RH . When thetc. Moreover, the accuracy of conversion formula, measurement circuits, and calculation equipment are important in the psychrometer measurement process. These factors combine to affect the measurement result and limit the widespread usage of the psychrometer.A psychrometer measures the relative humidity indirectly from two temperature probes. By the structure of the dry and wet bulb probes, the temperature sensing element is encased within a stainless steel tube. The sensing element is not in direct contact with the environment. The psychrometer avoids directly exposing the relative humidity sensing element to the measurement environment. This key difference allows the psychrometer to be used in difficult measurement environments. However, the relative humidity measurement process of the psychrometer is a complex physical process. Many factors can influence the measurement result such as barometric pressure, ambient temperature, velocity of the air flow, accuracy of temperature probes, location of the probes, wet cloth size and quality, et al. introduced a parameterized psychrometer coefficient with wind speed in their paper [Nakahama investigated the psychrometer construction for performance testing in temperature and humidity chambers and the accuracy of humidity measurements meeting the challenge of quality engineering . Nakahamir paper .From the above discussion and the literature, it is clear that the psychrometer has been widely used as a method for relative humidity measurement over a wide range of values in a harsh environment, with varying degrees of measurement accuracy. Therefore, it is important to study the interrelated elements in the measurement environment and optimize the measurement accuracy of the psychrometer. This paper introduces an improved measurement device of aspirating psychrometer with specific construction parameters and provided real test results that are conducted in a meat-drying room in a food company. A specific psychrometric measurement device was developed and tested, including the structure of the improved psychrometer, signal conversion method, calculating unit, and the real-time test results. The influences of the psychrometer structure and interrelated factors of the measurement environment were demonstrated. The research analyzed the accuracy, stability, and reliability of the specific improved psychrometric measurement method based on a real-time temperature and relative humidity process control system.In this section, a common psychrometer implementation is briefly given. Then, a method of signal conversion between the dry and wet temperature probe measurements and relative humidity that is suitable for digital processors is presented.We tested several temperature sensors, including the thermocouple temperature sensor and the resistance thermometer. The PT100 is a resistance thermometer showed better accuracy and consistency than the thermocouple temperature sensors. Furthermore, the thermocouple temperature and the resistance temperature have a strong hull of stainless steel and its ingress protection number is 67. Thus, we selected the PT100 as the dry and wet temperature probes for the improved aspirated psychrometer. In order to fit the special measurement environment, the probes were chosen to be stainless steel with waterproof cable connections like IP67 ingress protection. The wet bulb temperature probe was soaked in water via a piece of cotton cloth. A fan was used to allow consistent and controllable airflow over the probes. The dry bulb and wet bulb temperature are denote by As an indirect measurement method, signal conversion between temperature and relative humidity is an important factor that directly affects the measurement accuracy of the psychrometer. Prior references exist on different conversion methods ,36 and tThe signal conversion is based on the Sprung primary formula . The masThe ratio of the partial pressure of water vapour in the mixture to the saturated pressure of water vapour at a prescribed temperature is defined as relative humidity, normally expressed as a percentage byituation . Ps is tP is the station pressure, C is a constant. The value of C differs according to the measurement environment. When the ambient temperature is above freezing, C = 0.5; and when the ambient temperature is below freezing, C = 0.43 [The partial pressure of water vapour in the mixture can be calculated using the Sprung formula  by(3)PdC = 0.43 . In thisC = 0.43  is validC = 0.43 . In a NAC = 0.43 . The uniThe These two equations are tested to valid for meat drying processes when the temperature is the in range of 4–18 °C. Finally, the relative humidity Based on the measurement methods for relative humidity, the humidity sensors can be classified into two categories: direct and indirect measurement methods. The measurement principles of capacitive humidity sensor and resistive humidity sensor belong to the category of direct measurement methods. The indirect measurement methods of humidity do not expose the humidity sensitive device to the measurement environment. The psychrometric measurement method is a typical indirect relative humidity measurement method, which measure the temperatures and then convert them into RH values.This research focuses on the structural parameter’s improvement of the aspirated psychrometer and specifically analyzes the influence of several factors in the measurement accuracy of the aspirated psychrometer based on the practical measurement. The experiments that were conducted included: the air flow speed test that air flow is passing through the dry and wet bulb temperature probes; the cloth size test that is put on the wet bulb probe; and the distance test between the wet bulb probe and water level. A specific improved psychrometer device was implemented in order to perform these experiments. There are different styles of psychrometers, and for the same style of psychrometer, the construction sizes are mostly different. The motivation to select two different distances to demonstrate that the effect of the distance between the water level and the wet bulb cannot be ignored. The first experiment was set up with two different distances between the wet bulb probe and the water level, at 23 mm and 71 mm. The variable was the air flow speed, which was changed from 0 m/s (stationary) to 4 m/s at 0.5 m/s intervals. The size of the wet cloth was 63 mm × 122 mm. The test results are shown in The second experiment was designed to examine the influence of different cloth size to wet bulb temperature. In the experiment, the cloth size was changed to 126 mm × 122 mm and other variables were same as in the first experiment. The test result is shown in The influence of environmental factors is shown in Based on the above results, we estimated the sensitivity of wet bulb temperature to the variation of various environmental factors. At a constant air flow speed of 2 m/s, different cloth sizes caused 0.2 °C uncertainty in the wet bulb temperature. Different distances between wet bulb temperature probe to water level added 0.3 °C uncertainty, and if the air flow speed changed from 2 m/s to 0.5 m/s then the uncertainties would increase approximately 0.3 °C. The total change was approximately 0.8 °C of the measured wet bulb temperature value.From Based on the analysis in The dry bulb and wet bulb temperature signals were measured in a three-wire circuit using a specific integrated circuit-ASIC 7030 A/D for analog to digital conversion. The profile controller operated with a micro-controller to acquire the measurement data. The test instruments include A/D converter and micro controller. The ASIC 7030 was used as the A/D converter. The two styles of micro controllers used were Neuron chip 3150 BF by Toshiba Corporation Semiconductor Company  and 89 C52 by ATMEL Ltd. . Several accurate instruments were used to calibrate the specific improved psychrometer measurement device and measure its accuracy. The instruments included: VAISALA humidity and temperature probe (HMP75) and measurement indicator (MI70), PACER digital anemometer (DA400), Ebro precision thermometer (TFX 430), MadgeTech accurate data logger (RHTemp 101A) for temperature and relative humidity, and Fluke instrument dry-well (9102S).In these experiments, the dry-well (Fluke 9102S) and Ebro precision thermometer (TFX 430) were used for dry bulb and wet bulb temperature probe calibration. The accuracy of Ebro precision thermometer (TFX 430) is ±0.05 °C and resolution is 0.01 °C. The humidity and temperature probe (HMP75) and measurement indicator (MI70) were used as a confirmation instrument. The improved psychrometer and the humidity and temperature probe (HMP75) with measurement indicator (MI70) were tested in a drying room environment. The test results of the improved psychrometer and the humidity measurement instrument (MI70 with HMP75) are shown in The test data shows that the measurement value of the improved psychrometer is very close to the measurement value of the humidity measurement instrument (MI70 with HMP75). The deviation between the two instruments was less than ±0.5% RH. According to the certified accuracy of the humidity measurement instrument (MI70 with HMP75), the measurement accuracy of the improved psychrometer was ±0.6% RH in a drying room measurement environment.A comparison test was made between the original psychrometer and the improved psychrometer in a functioning meat drying room control environment. The original psychrometer device was removed from the same general drying room control system.The original psychrometer device and the improved psychrometer device used the same probe and instrument. The calibration instrument was the same as in the previous section, and the atmosphere pressure was 964 hPa at the time of the experiment.The test results for temperature and relative humidity are shown in The measurement accuracy of the original psychrometer and the improved psychrometer are clearly shown in Another issue of a relative humidity sensor is the stability of measurement signal. Most relative humidity sensors have zero drift issue which depends on the properties of the relative humidity sensitive element. This issue is more evident in a harsh environment such as the drying room for meat production. Measurement stability of the prototype psychrometer was quantitatively analyzed in a real-time temperature and relative humidity control system. The experiments in a drying room control system using the prototype psychrometer as the relative humidity sensor was conducted. The testing environment was challenging since the air contained smoke particles, which did not allow capacitive relative humidity sensors to work at any extended period of time. It is a big challenge to have a sensor work in meat-drying room environments to measure the relative humidity. Since the humidity sensitive device of capacitive humidity sensors or resistance humidity sensors is exposed in a measurement area, it cannot perform properly in a harsh environment. The real tests show that some relative humidity sensors, such as capacitive humidity sensors or resistance humidity sensor, do not work properly for a long period of a time. A psychrometer does not use the humidity sensitive device for relative humidity measurement. It uses two temperature probes, named the dry bulb and wet bulb probes, to measure the relative humidity indirectly. This feature of measurement demonstrates that the modified psychrometer cam work stably in a harsh measurement environment for a very long period of time.3 at −7 °C, and heating source of the system was low pressure steam at 15 PSI. The humidifier was a dry fog generator with water droplet size of ≤4.2 microns. The drying room control system was two independent closed-loop control systems for temperature and relative humidity using the proportional, integral, and differential control algorithm. The relative humidity sensor was a prototype psychrometer.The dimension of the test drying room was 28 m × 9 m × 3.8 m. The control variables were the room temperature and relative humidity. The cooling source of the system was NHTwo temperature and relative humidity data loggers (MadgeTech TempRetriever-RH and RHTemp101A) were used as the recording measurement equipment. Among them, the TempRetriever-RH had a relative humidity resolution of 0.1% RH and a temperature resolution of 0.1 °C. The RHTemp101A had a relative humidity resolution of 0.1% RH and a temperature resolution of 0.01 °C. The set points of temperature and relative humidity for the drying room control system were 13 °C and 74% RH. The test results of temperature and relative humidity under closed-loop control are shown in The 65 h recording of temperature and relative humidity in a drying room under closed-loop control is shown in Temperature directly influences relative humidity measurement accuracy and stability. The effect of the varying factors in the relative humidity measurement can be evaluated using Based on The test results showed the temperature control accuracy was achieved ±0.05 °C for over seven hours in the control system. In The above test results distinctly revealed the influence of temperature control accuracy to the relative humidity control accuracy in the drying room control system. When the temperature control accuracy was ±0.1 °C, the relative humidity control accuracy was ±0.8% RH , and wheThis paper focused on a discussion of the application for the relative humidity measurement device in a current control system in challenging environments, in particular, the drying system for dry-cured meat products. Test results revealed the effects of different parameters of the psychrometer in the measurement accuracy and stability of relative humidity.The relative humidity measurement accuracy of the specific improved psychrometer that can be achieved is ±0.6% RH in a meat manufacturing environment.The specific improved psychrometer can perform long-term, stable, and reliable work in a severe environment of a meat manufacturing drying room.Based on the test results of a drying system for dry-cured meat products using a specific improved psychrometer, the control accuracy of a relative humidity system that can be achieved is ±0.8% RH.Based on experimental results, the characteristics of the specific improved psychrometer are summarized as follows.In summary, these characteristics of the improved psychrometer, including its higher measurement accuracy and stability, make the psychrometer especially suitable as a relative humidity sensor for applications in challenging measurement environments.


Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated.Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ≥95% identity, with one example sequence chosen to be the representative.These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate. Although not numerous, group II introns are found widely across the domains of life, being present in eubacteria, archaebacteria, and eukaryotic organelles -3. The cThe retromobility mechanism of group II introns has been well-studied biochemically and genetically, and thorough descriptions can be found elsewhere ,7,12,13.Approximately 25% of eubacterial genomes and 10% of archaeal genomes contain at least one group II intron or a fragment of an intron ,17. NearGroup II introns can be classified either phylogenetically according to their IEPs, or by features of their RNA secondary structures. Based on the IEPs, eight lineages of group II introns have been identified  ,19. By cThe IEPs of group II introns are easily identified through BLAST searches, because of their conservation. Incomplete IEPs are identified as truncations, in cases where RT subdomains or the X domain are missing or have premature stop codons. By contrast, the RNA component is not easily identified due to insufficient sequence conservation. For many introns, the RNA must be folded into a secondary structure in order to confidently predict the correct boundaries. Identifying the RNA boundaries is critical given that they specify the splice sites and define the protein translation product.Across all group II introns, only domain V is highly conserved and reliably identified, while the remainder of the RNA has short and scattered segments of conservation . For exaA useful fact in identifying RNA boundaries is that the ribozyme and IEP co-evolved  . Thus, tIn the past we have compiled and catalogued group II introns in bacterial genomes in order to define structural types, distribution and spread, and to collect data for evolutionary analyses . HoweverThe pipeline consists of eleven programs listed in Table To briefly summarize the process, the 22 representative IEPs are used as queries to search GenBank for candidate IEPs. Detected DNA sequences are downloaded along with 3 kb of flanks upstream and downstream of the IEP. The putative IEPs are screened to retain only bacterial and archaeal sequences, and to exclude RTs that are not group II introns. Each candidate intron is tentatively assigned to a class . The ORF domains present in each IEP are determined and IEPs that are judged to be intact are assigned an amino acid sequence. Intron RNA boundaries are predicted based on class-specific sequence profiles for 5′ and 3′ boundaries. To eliminate redundancy in the final set of introns, introns are grouped into sets of ~95% identity and one intron in each set is selected as the representative, or prototype.The output of the programs is in two folders, one containing data for the complete set of full-length and presumably functional introns, and the second containing data for a reduced set of non-redundant introns (<95% identity); however, if an intron in a 95% group is from a different species, it is included as a unique intron despite the sequence redundancy. In addition to this final output, data can be accessed at each step of the pipeline, and it is possible to write the accumulated data to more reader-friendly files using flags.http://hmmer.janelia.org/software/archive; the path to the HMMER2 programs should be an environment variable $HMMER2), HMMER3 ; the BLAST + suite (2.2.26) from NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.26/); CLUSTALW ; and PHYLIP (3.69/ http://evolution.genetics.washington.edu/phylip/getme.html). Other modules such as BioPerl utilities are embedded within the executable files.The programs run on a 64-bit LINUX system with internet access. Programs that must be installed on the computer include the following: HMMER2  where updates will become available.The program runs by command lines. Typical commands are specified in Additional file -20. The use of TBLASTN is important because it detects unannotated IEPs and the output format gives genomic coordinates of the ORF matches. If desired, the user can choose to use another set of ORFs as queries, or search another GenBank database , or specify a different E value cut-off . For each ORF, the program connects to NCBI and searches GenBank for sequence matches using TBLASTN (protein query vs. translated DNA database), and using the non-redundant database with an E value cut-off of 1x10For each accession number and coordinates, the DNA sequence is downloaded from GenBank in standard GenBank format along with 3 kb of flanking sequence both upstream and downstream of the HSP match . The DNA sequence is downloaded in the orientation that encodes the IEP on the forward strand. Taxonomic information is collected from the GenBank entry, which allows the files to be divided into five sets: “bacteria & archaebacteria”, “eukaryotes”, “mitochondria”, “chloroplasts”, and “other.” For each of the five sets, the texts of all of the GenBank entries are concatenated and saved as a single text file. The “bacteria_and_archaea” set is used by default in subsequent programs, but it is possible to input other folders or to introduce other folders as input of data in subsequent programs.A folder is created, “storage_new”, which over the course of the pipeline will contain a series of accumulated information. At this point, the folder “0” is created, but in subsequent programs storage folders 1–7 are generated, each containing progressively more data as further analyses are done Figure . Folder A significant challenge in assembling a comprehensive set of group II introns is that there are RTs in bacteria that give BLAST matches to IEPs but are not group II introns. One way to eliminate them is to use a more stringent E value cut-off in the initial BLAST search; however, the stringency also eliminates some true group II introns that are not closely related to those already identified. Complicating this scenario is the fact that some RT types are very closely related to group II introns and presumably derived from them, yet do not have associated RNA structures  . Therefo-20 in the BLAST search; relaxing the stringency mainly increases the number of short fragments of group II introns. Previously, we compiled a list of RT types in bacteria, including group II introns, retrons, diversity-generating retroelements and a variety of additional uncharacterized RTs [Our strategy to deal with this issue is to do multiple BLAST searches with relatively low stringency, using as queries a variety of intron subtypes, and then combining the results and subtracting out the non-group II introns. We use an E value cut-off of 1x10ized RTs . Using tThe phylogenetic class for each candidate IEP is next assigned, using a BLASTX search (translated nucleotide query vs. protein database) in which a local database of 475 group II intron IEPs is searched using as a query each candidate DNA sequence. If the three best matches are of the same class, the candidate is taken to belong to that class as well. Classes assigned are ML, CL, A, B, C, D, E, F, undefined, or unclassified. “Undefined” denotes that the top three hits are not of the same class, whereas “unclassified” means that the top three hits are to introns designated “unclassified”). The BLAST results are also used to identify the closest intron relative of each candidate among the 475 curated group II introns, which will be used later in the pipeline.To determine which ORF domains are present in each candidate sequence, a set of nine reference IEPs is used, with one IEP per class . For each reference IEP, one conserved amino acid in each domain  is chosen as a proxy for the presence of that domain. A BLASTP alignment is made between each candidate sequence and the reference intron for its class. If a given amino acid proxy is present in the pair-wise alignment, then the IEP is judged to contain that domain . This step determines whether the candidate contains all expected IEP domains, or is truncated and missing expected domains. Candidate sequences are sorted into folders according to the ORF characteristics. The IEP is assigned as “normal” if all domains are present once in the sequence, “missing domains” if one or more expected domains are absent, “extra domains” if one or more domains are present more than once , and “missing and extra domains” if one or more domains are missing and one or more domains are present more than once . By default, only complete IEPs continue to the next step in the pipeline.The presence of all domains does not necessarily mean that the IEP is intact, because there may be frame shifts, premature stops, or other problems within the ORF. While predicting IEP function is inherently speculative, we use the criterion of a single, uninterrupted alignment between a candidate IEP and its closest, curated IEP relative. To identify such proteins, a pairwise BLASTX alignment is done between each candidate DNA sequence and its closest IEP relative. A putatively intact ORF is indicated by the absence of stop codons, by the absence of insertions (gaps), and also by the alignment consisting of a single HSP . If no problems are identified, then the amino acid sequence of the IEP is assigned. Because many GenBank sequence files are not annotated, and also because N-termini are often misannotated in GenBank, the IEP sequences are assigned based on the amino acid sequences of the closest relative in the set of 475 curated introns. The start codon is chosen according to the start codon of the closest annotated group II intron sequence and the presence of an upstream Shine-Dalgarno sequence.As described above, group II intron boundaries are not identified reliably on the basis of a generalized consensus sequence; however, class-specific sequence patterns give greater accuracy. Taking this into account, hidden Markov model (HMM) profiles have been made for the boundary sequences of each class, and are used to search for potential 5′ and 3′ termini. Simple consensus sequences of the boundary sequences for the classes are shown in Additional file The profile matches for each intron candidate are evaluated in order to judge the most probable intron boundaries out of the compiled possibilities. A full-length, intact intron is indicated when there is only one plausible 5′ and one 3′ boundary, and they are in the correct order and separated by a reasonable distance. If these conditions are met, then the full intron sequence is generated based on those boundaries. Candidate introns are sorted into folders of “have boundaries” and “ambiguous intron boundaries”. By default, only introns with “have boundaries” continue to the following programs.Because group II introns are mobile DNAs, there are sometimes many identical or nearly identical intron sequences in a genome or in different GenBank entries. In order to eliminate the redundancy, introns are grouped into sets of introns of >95% identity. To do this, each phylogenetic class of IEPs is analyzed separately. The ORF sequences are aligned across the region corresponding to domains 0 to X, using ClustalW . The aliFinally, one intron in each group of 95% identity is chosen to be the representative, or prototype, unless there are multiple species within the group, in which case each species is assigned a prototype as well. The final output goes to a series of folders and files listed in Additional file The described programs successfully collect and download sequence information from GenBank, sort them into classes, identify the ORF and ribozyme components and boundaries, and create a non-redundant list with <95% identities. At each step of the pipeline, an aspect of the introns is examined and introns appearing not to be full-length and/or functional are set aside, with only intact sequences going forward. This produces a steadily smaller set of introns, with more information accumulated at each step for the introns that remain. As of July 2013, 3,191 non-redundant HSPs were identified initially as candidate group II introns in the “blast_and_parse” step, while at the end of the pipeline 572 of these were identified as prototypes. A detailed account of the segregation of the 3,191 sequence files into different categories over the course of the programs is listed in Additional file To measure the accuracy and completeness of the pipeline, a set of 513 introns present in the initial set of 3,191 downloaded sequences was followed through the process. At the end of the pipeline, 451 of 513 (88%) were included in the set of prototypes, which by default includes only “high probability” introns. If introns in the categories of medium and low probability are included as well, then 479 of 513 (93%) were identified. If one considers only the single step that identifies boundaries for introns using the HMM profiles (“find_intron_boundaries”), and take into account the best predictions regardless of the probability estimates , then 477 out of 492 (97%) introns analyzed by the program have the correct boundaries.A substantial portion of the missed introns  corresponds to the set of twintrons (introns nested within other introns) and other candidate DNA sequences containing more than one intron copy. These sequences were excluded at the “find_orf” stage of the pipeline. Their removal serves to prevent errors in subsequent steps due to multiple introns being present in a sequence being analyzed; however, it has the consequence of excluding sequences with more than one intron in the 8–10 kb of downloaded sequence. These introns, however, can be recovered from the “multiple ORF locations” folder generated by the “find_orf” program, and manually examined to determine their exact organizations.A major reason for the remaining missed introns is the specificity of some of the HMM profiles. Sequences considered “unclassified” were screened with the profiles of other classes, which not surprisingly reduced specificity and success. In addition, boundary profiles for Classes E and F were constructed from a relatively small number of introns and the prediction success was notably lower than for profiles of other classes. Overall, the boundaries for Class E, F and unclassified introns were correctly predicted with “high probability” for only 36 of 67 introns (54%) by the program “find_intron_boundaries.” However, including the single, best “low” or “medium” probability prediction resulted in 58 of 67 introns (87%) with correct boundaries.In the future, the HMM profiles can be improved substantially by increasing the number of E and F introns in the alignments, and by identifying additional classes from the “unclassified” introns, when enough examples are available to define a group and corresponding sequence pattern. An alternative strategy for identifying introns not belonging to established classes is to manually examine the sets of failed sequences after the “find_intron_boundaries” program. These sequences have intact IEP sequences but lack predicted 5′ and/or 3′ boundaries. Several of them appear to be bona fide introns that belong to new classes (unpublished data), which will be reported elsewhere.Another limitation of the boundary prediction algorithm is illustrated by two sets of introns that have insertions or extensions at either the 5′ or 3′ termini. An unusual subset of CL1 (IIB1) introns has a 5′ insertion near the start of the intron, which can be hundreds of nucleotides long . A seconThiomargarita namibiensis. However, these introns do not appear to be common in bacteria [Finally, it should be noted that introns without IEPs will be missed by this algorithm, as well as introns that encode an IEP not belonging to the RT family, such as the LAGLIDADG-encoding intron in bacteria .This suite of programs allows for comprehensive, automated detection of group II introns from GenBank, and provides an alternative to manual curation of group II introns amidst the rapidly expanding sequence databases. While not without limitations, the programs give effective tools for handling group II intron sequences and determining the scope and diversity of group II intron sequences present in bacterial genomes. Future updates to the programs, as well as larger libraries of curated group II introns, will improve the performance of the pipeline over time.• Project Name: Group II intron identification pipelinehttp://webapps2.ucalgary.ca/~groupii/index.html• Project Home Page: • Operating System: Linux 64-bit• Other requirements: HMMER2 2.3.2, HMMER3 3.0, BLAST + Suite 2.2.26, ClustalW 2.1, PHYLIP 3.69• License: None• Restrictions of use by non-academics: NoneD: DNA binding domain; En: Endonuclease domain; HMM: Hidden Markov model; IEP: Intron-encoded protein; RNP: Ribonucleoprotein; RT: Reverse transcriptase.The authors declare that they have no competing interests.Programs were written by MA, MAC, AD, KSH, TL, RAEN, AS, and RS, and were devised by these authors and SZ. Output generated by the programs were evaluated by LW and AMJ. The manuscript was written by SZ and CJS with input from all authors. All authors read and approved the final manuscript.Class-specific consensus sequences for 5′ and 3′ intron boundaries.Click here for fileTable S2. Output of each program.Input commands and options for each program. Click here for file