TLR2, TLR4 and TLR9 genes, in the development of human cytomegalovirus (HCMV) infection in pregnant women and their fetuses and neonates, was performed.Human cytomegalovirus (HCMV) is the most common cause of intrauterine infections worldwide. The toll-like receptors (TLRs) have been reported as important factors in immune response against HCMV. Particularly, TLR2, TLR4 and TLR9 have been shown to be involved in antiviral immunity. Evaluation of the role of single nucleotide polymorphisms (SNPs), located within TLR2 2258 A > G, TLR4 896 G > A and 1196 C > T and TLR9 2848 G > A SNPs were determined by self-designed nested PCR-RFLP assays. Randomly selected PCR products, representative for distinct genotypes in TLR SNPs, were confirmed by sequencing. A relationship between the genotypes, alleles, haplotypes and multiple variants in the studied polymorphisms, and the occurrence of HCMV infection in pregnant women and their offsprings, was determined, using a logistic regression model.The study was performed for 131 pregnant women, including 66 patients infected with HCMV during pregnancy, and 65 age-matched control pregnant individuals. The patients were selected to the study, based on serological status of anti-HCMV IgG and IgM antibodies and on the presence of viral DNA in their body fluids. Genotypes in P > 0.050). GG homozygotic and GA heterozygotic status in TLR9 2848 G > A SNP decreased significantly the occurrence of HCMV infection . The G allele in TLR9 SNP was significantly more frequent among the uninfected pregnant women than among the infected ones . Considering other polymorphisms, similar frequencies of distinct genotypes, haplotypes and multiple-SNP variants were observed between the studied groups of patients.Genotypes in all the analyzed polymorphisms preserved the Hardy-Weinberg equilibrium in pregnant women, both infected and uninfected with HCMV (TLR9 2848 G > A SNP may be associated with HCMV infection in pregnant women.The online version of this article (doi:10.1186/s12985-017-0730-8) contains supplementary material, which is available to authorized users. Human cytomegalovirus (HCMV) is the most common cause of intrauterine infections worldwide, with seroprevalence rates at the range from 40 to 100% among pregnant women –7. In cain vitro study, TLR2 was found as a target of HCMV miR-UL112-3p [Taking into account the immune response to HCMV, the Toll-like receptors (TLRs) have been reported to play important role –15. PartUL112-3p . PreviouUL112-3p . In turnUL112-3p . In humaUL112-3p . A studyUL112-3p .TLR genes, was also reported [TLR2 + 1350 T > C polymorphic site, the CC genotype  was correlated with congenital HCMV infection [TLR2 + 1350 T > C as well as 2029 C > T SNPs among the analyzed Polish fetuses and neonates, who were both congenitally infected and uninfected with HCMV [TLR2 2258 G > A SNP to be significantly more frequent among the infected offsprings than among uninfected individuals [TLR2 2258 SNP was shown to be associated with TLR2 signaling impairment [TLR4, both 896 A > G and 1196 C > T polymorphisms were reported to impair TLR4/MD2 dimerization necessary to activate downstream signaling, involved in HCMV-induced immune response [TLR4 896 A > G and TLR4 1196 C > T SNPs were also determined to be significantly associated with more frequent opportunistic infections and cytomegaly, diagnosed among renal transplant recipients (RTRs) [TLR4 SNPs to be possibly associated with the risk factors of invasive aspergillosis that included HCMV seropositivity [TLR9, the -1237 T > C SNP was marginally correlated with recurrent urinary infections in RTRs [TLR4 1196 polymorphism, as well as the GA variant in TLR9 2848 G > A SNP to be correlated with the infection, and the heterozygotic status in TLR9 SNP increased the risk of congenital cytomegaly by 4.81 times [TLR2 2258 and TLR9 2848 G > A polymorphisms, were found to be associated with an increased risk of congenital HCMV infection [TLR9 -1486 T > C and 2848 C > T SNPs, the heterozygous and homozygous recessive genotypes within the reported polymorphisms, were associated with an increased risk of HCMV disease among infants [Previously, the role of single nucleotide polymorphisms , residing within reported –26. In cnfection . In turnith HCMV . Howeverividuals . In an iresponse . TLR4 89s (RTRs) , 31. Anositivity . Conside in RTRs . The stu81 times . Moreove infants .TLR2 2258 G > A, TLR4 896 A > G and TLR4 1196 C > T, as well as TLR9 2848 G > A SNPs and the occurrence of HCMV infection among pregnant women seems to be really possible, although there have been no such reports. Therefore, the current paper was aimed to describe the role of TLR2, TLR4 and TLR9 SNPs , ventriculomegaly , respiratory failure , heart defects , hepatitis , ascites , intrauterine growth restriction , as well as fetal/neonatal death , and were observed among the 28.8% (19/66) offsprings of the infected pregnant women. Active HCMV infections were confirmed in 15 pregnant women  and their 10 offsprings , based on the presence of the viral DNA in body fluids, including maternal whole blood, plasma, and urine samples, as well as fetal amniotic fluids and umbilical cord blood specimens. Considering all the studied parameters related to HCMV infection, the overall rate of congenital viral transmission from mother to the fetus was 33.3% (22/66). The study was approved by the Research Ethics Committee at the Polish Mother’s Memorial Hospital—Research Institute. All the samples, previously collected for diagnostic purposes, were anonymized in this report. Informed consent forms were signed by all the enrolled pregnant women.The study was performed with 131 pregnant women, including 66 patients infected with HCMV during pregnancy, and 65 age-matched control individuals uninfected with the virus, at the age between 18 and 41 years (mean 28.52 years). The mean age among HCMV-infected pregnant women was 28.29 ± 5.29 years, and among uninfected pregnant women was 28.75 ± 4.79 years. The samples were obtained from pregnant women, admitted to the Department of Feto-Maternal Medicine and Gynecology at the Polish Mother’s Memorial Hospital—Research Institute, between the years 2002 and 2014. Clinical samples, used in the oC until analysis, on the day of blood collection. Serological status was determined at the Hospital’s Department of Clinical Microbiology.Blood specimens were gained from the pregnant women by venipuncture on their first visit to the Hospital, between the 5th and 38th week of gestation (mean 21.96 weeks). The mean gestational age among HCMV-infected pregnant women was 22.50 ± 9.21 weeks, and among uninfected pregnant women was 21.63 ± 9.02 weeks. Serum samples were obtained by centrifugation and then stored at 4Screening for anti-HCMV IgG and IgM antibodies was performed with VIDAS CMV IgG and IgM tests , used between the years 2002 and 2006. Both IgG and IgM antibodies titers ≥ 6 IU/ml were determined as positive. Between 2006 and 2011, the antibodies levels and IgG avidity, were evaluated by the chemiluminescence immunoassays (CLIA), using anti-CMV IgG and IgM tests . Samples were considered as IgG- or IgM-positive for antibody levels ≥ 0.6 IU/ml and ≥ 30 IU/ml, respectively. The IgG avidity with indexes < 0.200 was interpreted as low, 0.200–0.300 as borderline, and ≥ 0.300 as high. From the year 2012, the CLIA method was replaced by the enzyme-linked fluorescence assays (ELFA), used to determine antibodies levels. IgG antibody titers ≥ 6 IU/ml and IgM indexes ≥ 0.9 were considered positive.oC until further genetic analyses, according to the manufacturer’s guidelines.Genomic and viral DNA was extracted from studied body fluids, using a QIAamp DNA Mini Kit . The isolated DNA was diluted in 100 μL of elution buffer and stored at -20UL55 gene fragment, as described previously [5 to 1 plasmid DNA, containing the entire HCMV UL55 open reading frame [Among HCMV infected patients, the amount of viral DNA in the study specimens, was determined by the quantitative real-time PCR assay for the detection of eviously –42. The ng frame . The ampTLR2 2258 G > A, TLR4 896 A > G and 1196 C > T SNPs, as well as TLR9 2848 G > A SNP , to verify the genotypes, determined by the PCR-RFLP assay. Sequencing was performed for five GG homozygotes and seven GA heterozygotes in TLR2 SNP, for six AA homozygotes and four AG heterozygotes in TLR4 896 A > G SNP, for five CC homozygotes and five CT heterozygotes in TLR4 1196 C > T SNP, as well as for five GG homozygotes, five GA heterozygotes and five AA homozygotes in TLR9 SNP. The exemplary chromatograms for DNA fragments of sequenced PCR products encompassing different TLR SNPs are presented in Additional file TLR genes were compared, using the BLASTN program for the alignment of two (or more) sequences . Chromatograms were read, using the Chromas Lite 2.1.1 program.ly shown , 28, 44.escribed –49; see TLR2, TLR4 and TLR9 SNPs among HCMV-infected and uninfected pregnant women was estimated by means of descriptive statistics. The studied groups of patients were tested for the Hardy-Weinberg (H-W) equilibrium, the linkage disequilibrium (LD) and haplotypes, using the SNPStats software (http://bioinfo.iconcologia.net/en/SNPStats_web). Genotypes in all the analyzed polymorphisms preserved the H-W equilibrium in pregnant women, both infected with HCMV and control uninfected individuals (P > 0.050). The TLR4 896 A > G and 1196 C > T SNPs were in linkage disequilibrium among the studied groups of pregnant women (P ≤ 0.050). The relationships between the genotypes, alleles or haplotypes in TLR SNPs and the occurrence of the viral infections were determined by cross-tabulation, Pearson’s Chi-squared test and by the logistic regression model. The multiple-SNP analysis for the haplotypes in TLR4 SNPs, as well as for the complex genotypic variants within the range of all the analyzed TLR SNPs was performed by the Expectation Maximization (EM) algorithm. The outcomes of the analyses were determined statistically significant when the significance level of P ≤ 0.050 was obtained. The statistical analysis was in part supported by the NCSS 2004 software.Distribution of genotypes and alleles in the analyzed In pregnant women, HCMV infection during pregnancy was estimated on the basis of IgG seroconversion within gestation, on the presence of IgG and IgM specific antibodies or on a low IgG avidity index. IgG seropositivity was confirmed in 96.55% (56/58) of the infected pregnant women, while data on IgM antibodies suggestive of the recent infection, were obtained for 86.21% (50/58) of infected individuals. Control group in the study, consisted of pregnant women seronegative for both IgG and IgM antibodies against HCMV, who were classified as uninfected individuals.2 copies/ml and ranged from 1.3 × 102 to 1.1 × 103 copies/ml, while the mean viral load was 5.2 × 102 copies/ml. In case of plasma specimens, the median viral load was 5.6 × 102 copies/ml, ranging from 1.8 × 102 to 3.6 × 103 copies/ml, whereas the mean viral load was 1.4 × 103 copies/ml. Regarding urine samples, the median HCMV DNA load was 4.4 × 102 copies/ml, ranging from 1.5 × 102 to 2.4 × 103 copies/ml, and the mean viral load was 3.2 × 103 copies/ml. Considering fetal amniotic fluids, the median HCMV DNA load was 9.9 × 102 copies/ml, and ranged from 2.2 × 102 to 1.5 × 103 copies/ml, while the mean viral load was 9.2 × 102 copies/ml. In umbilical cord blood samples, the median HCMV DNA load was 3.2 × 103 copies/ml, and ranged from 1.6 × 102 to 6.6 × 103 copies/ml, and the mean viral load was 3.3 × 103 copies/ml.The median load of HCMV DNA in whole blood specimens of the infected pregnant women was 3.8 × 10TLR2 2258 G > A polymorphic site were 93.9% (62/66) and 6.1% (4/66), respectively  and 8.2% (5/61) of the pregnant women, respectively, while CC and CT genotypes in 1196 C > T polymorphism—by 90.9% (60/66) and 9.1% (6/66), respectively. Considering TLR9 2848 G > A SNP, the GG, GA and AA genotypes were determined in 13.3% (8/60), 41.7% (25/60), and 45% (27/60) of patients, respectively. Among uninfected pregnant women, the frequencies of GG and GA genotypes in TLR2 SNP were 92.2% (59/64) and 7.8% (5/64), respectively. The frequencies of AA and AG genotypes in TLR4 896 A > G were 86.9% (53/61) and 13.1% (8/61), respectively, while the rates of CC and CT genotypes in TLR4 1196 C > T were 87.3% (55/63) and 12.7% (8/63), respectively. In cases of TLR9 2848 G > A polymorphism, the GG, GA and AA genotypes were observed in 20.3% (13/64), 53.1% (34/64), and 26.6% (17/64), respectively. Taking that into account, the GG homozygotic and GA heterozygotic status decreased significantly the occurrence of HCMV infection  and 3.0% (4/132), respectively  and 4.0% (5/122) of the patients, respectively, while C and T alleles in 1196 C > T SNP—in 95.0% (126/132) and 5.0% (6/132), respectively. Regarding TLR9 2848 G > A SNP, G and A alleles were carried by 34.0% (41/120) and 66.0% (79/120) of the infected pregnant women, respectively. Among uninfected pregnant women, the frequencies of G and A alleles in TLR2 2258 G > A SNP were 96.0% (123/128) and 4.0% (5/128), respectively. The frequencies of A and G alleles in TLR4 896 A > G SNP were 93.0% (114/122) and 7.0% (8/122), respectively, and the rates of C and T alleles in TLR4 1196 C > T SNP were 94.0% (118/126) and 6.0% (8/126), respectively. Considering TLR9 SNP, the G and A alleles were found in frequencies of 47.0% (60/128) and 53.0% (68/128), respectively. The G allele in TLR9 SNP was significantly more frequent among the uninfected pregnant women, as compared to the infected ones .Both GG homozygous and GA heterozygous variants in TLR9 2848 G > A SNP between the infected pregnant women analyzed in the current study, and the reported European populations might be due to a selection bias. Similarly, significantly higher prevalence rates of anti-HCMV IgG and IgM antibodies determined among the infected pregnant women studied in this paper, as compared to the previously reported cohort of Polish pregnant women between 2010 and 2011, as well as to other European populations, are possibly also caused by a classification of the patients to the presented study [TLR9 2848 G > A SNP in the occurrence of HCMV infection in pregnant women. Another research reported before that the GA heterozygotic status in the analyzed TLR9 SNP, carried by fetuses and neonates, congenitally infected with HCMV, had significantly increased the risk of the infection [TLR9 SNP was more frequent among the infected offsprings than among the uninfected ones, although that difference was statistically non-significant [TLR9 SNP in the occurrence of HCMV infection may have been age-related, as well as may have resulted from the classification bias. The qualification criteria of the pregnant women to the study included their serological status, the presence of HCMV DNA in their body fluids and were also based on confirmed congenital infection with the virus in their fetuses. In turn, the fetuses were diagnosed as congenitally infected with HCMV only on the basis of ultrasound markers and on the presence of HCMV DNA in their body fluids, since the diagnostics towards the infection is not routinely performed in pregnant women. Considering the role of TLR9 SNP in HCMV infection, the TT homozygotic status in TLR9 -1237 T > C SNP was reported to have been correlated with a decreased risk of the infection in HCMV-seropositive kidney transplant recipients [TLR9 -1237 T > C, as compared to the patients, carrying the T allele [TLR9 2848 G > A polymorphism was not involved in the incidence of HCMV infection among the examined patients [TLR9 -1486 T > C and 2848 C > T SNPs were associated with an increased risk of HCMV disease among infants [TLR9 gene, may have been correlated with various disease types. Regarding the HCMV infection, previous papers showed some contribution of TLR9 to its occurrence [TLR2 and TLR4 SNPs. The frequencies of the genotypes in the analyzed TLR2 and TLR4 polymorphic sites, found among HCMV-infected pregnant women were similar to the frequencies observed among European populations . Taking into account the allelic variability within TLR2 2258 G > A and TLR4 1196 C > T SNPs, the frequencies observed among all pregnant women studied in our research, were similar to the frequencies reported for European populations. In case of TLR4 896 C > T SNP, similarity to the European populations was found for HCMV-infected pregnant women (see https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986790). So far, no other study has shown before any possible involvement of TLR2 2258 G > A, TLR4 896 A > G and TLR4 1196 C > T SNPs in the occurrence of HCMV infection during pregnancy. However, in case of congenital HCMV infection, GA heterozygotic status and A allele within TLR2 2258 G > A, as well as CC genotype in the other TLR2 + 1350 T > C SNP, was reported to be correlated with the infection [TLR2 2258 G > A polymorphism to HCMV infection seems to be quite possible. Considering TLR4 SNPs, the CC genotype in TLR4 1196 C > T SNP, GC haplotype in both analyzed TLR4 SNPs, as well as GCA multiple variants within the range of TLR4 and TLR9 2848 G > A SNPs, were found to have been correlated with congenital HCMV infection in fetuses and neonates [TLR4 SNPs were similar to those, determined among congenitally infected fetuses and neonates, although no CT genotype at TLR4 1196 C > T polymorphic site was observed among the infected offsprings [TLR2 and TLR4 SNPs seems to be the important cause of the lack of any associations with HCMV infection. Similarly to our study, almost the same frequencies of distinct genotypes in both TLR2 2258 G > A and TLR4 896 G > A SNPs were determined in patients with transplants, with and without clinical signs of HCMV infection [TLR9 2848 G > A SNP seems to be the major polymorphism, contributing to HCMV infection in pregnant women during pregnancy. However, the studied genetic alteration is not involved, either in amino acid changes in TLR9 molecule or in regulatory site modifications within TLR9 gene [Differences in distribution of both the genotypes and alleles within ed study . So far,nfection . In turnnificant . The discipients . In reciT allele . On the patients . However infants . Therefocurrence , 23. In currence . The inhcurrence . What iscurrence , 53, 54.currence . The expcurrence . Considenfection , 28. Regneonates . In pregfsprings . Among pLR9 gene . TherefoTLR9 2848 G > A SNP seems to be involved in the occurrence of HCMV infection in pregnant women. Since both the GG homozygotic and GA heterozygotic statuses, as well as the G allele at TLR9 polymorphic site were significantly more frequent among the uninfected pregnant women, when compared with the infected ones, the genetic alterations in the studied polymorphism may plausibly be associated with the infection with HCMV. However, we suggest that further detailed studies would highly be justified to investigate the molecular mechanism of the TLR9 2848 G > A SNP function in HCMV infection.The outcomes presented in the current study show that Additional file 1: Figure S1.TLR2 2258 G > A (A), TLR4 896 A > G (B), TLR4 1196 C > T (C) and TLR9 2848 G > A (D) SNPs. DNA fragments were resolved in 2% agarose gel, stained with ethidium bromide. Disparate lanes show restriction profiles for distinct genotypes in the range of studied TLR polymorphisms, determined in different pregnant women. The numbers on the right side of electropherograms show the size of separated DNA fragments. M—50 bp DNA marker; GG, GA, AA, AG, CT, CC—genotypes determined in studied TLR polymorphisms. (TIF 501 kb)Exemplary PCR-RFLP products representative for various genotypes within Additional file 2: Figure S2.TLR2 2258 G > A , TLR4 896 A > G , TLR4 1196 C > T  and TLR9 2848 G > A (G-I) SNPs. For TLR2 SNP, DNA forward strands were sequenced, and for TLR4 and TLR9—reverse strands were assayed. The numbers above some peaks of chromatograms indicate the following nucleotides determined in sequenced DNA fragments. Loci of the polymorphisms and genotypes analyzed in the study, are indicated with arrows. GG, GA, AA, AG, CT, CC—genotypes determined in studied TLR SNPs. (ZIP 1732 kb)a and b Representative chromatograms of DNA fragments of various sequenced PCR products, encompassing


P-value) and odd ratio as well as cohort information such as size and ethnicity. Our evaluation illustrates that SNPcurator was able to replicate a large number of SNP-disease associations that were also reported in the NHGRI-EBI Catalog of published GWASs. SNPcurator was also tested by eight external genetics experts, who queried the system to examine diseases of their choice, and was found to be efficient and satisfactory. We conclude that the text-mining-based system has a great potential for helping researchers and scientists, especially in their preliminary genetics research. SNPcurator is publicly available at http://snpcurator.science.uu.nl/.The uniqueness of each human genetic structure motivated the shift from the current practice of medicine to a more tailored one. This personalized medicine revolution would not be possible today without the genetics data collected from genome-wide association studies (GWASs) that investigate the relation between different phenotypic traits and single-nucleotide polymorphisms (SNPs). The huge increase in the literature publication space imposes a challenge on the conventional manual curation process which is becoming more and more expensive. This research aims at automatically extracting SNP associations of any given disease and its reported statistical significance (Database URL: http://snpcurator.science.uu.nl/ Ever since its completion in 2003, the Human Genome Project has accelerated and encouraged research on decoding the genome structure and functionality, powered by the huge advances in the genotyping technologies. The main goal of genomic studies is to identify and reveal the genetic variations associated with diseases and its prevalence across different populations. Such studies contribute to more tailored detection, prevention and treatment of diseases which lay the groundwork for the era of personalized medicine . In the By definition, a SNP is a single base-pair  variation that occurs at a specific site in the DNA sequence. It does not directly cause a disease but increases the genetic predisposition of individuals towards a certain disease and can affect their responses to drugs and medications . CurrentText-mining tools have been employed recently to overcome the mentioned limitations and accelerate the curation process. Mutation finder (MF) extracts mutations through regular expressions while tmVar  also extMost of the above tools achieved high performance levels on different corpora. However, there is still a gap between the research community and the biomedical text-mining community. Yepes and Verspour compare in  the perfIn this article, we present the SNPcurator, a system more oriented towards information extraction specifically in the genome wide and candidate genes studies. The proposed model is constructed out of different natural language processing (NLP) modules to aid scientists in their search for relevant disease-associated SNPs through an intuitive web interface. It incorporates both syntactic and semantic methods to extract relevant information from PubMed abstracts such as cohort size and ethnicity, SNP ids and the reported results. The motivation behind this research is to create a publically available, scalable and fully automated extraction tool.http://snpcurator.science.uu.nl/ and allows researchers to easily query and search diseases and provides a useful resource for overview and summarization of associated SNPs found in literature. Sample code and the files used for evaluation are also available for download. SNPcurator has an online web interface at https://eutils.ncbi.nlm.nih.gov/entrez/eutils/), in particular Esearch and Efetch, are used in conjunction with the BioPython module to query the PubMed database. All articles included in PubMed are associated with Medical Subject Headings (MeSH) terms (http://www.ncbi.nlm.nih.gov/mesh). MeSH is a controlled vocabulary used for indexing articles according to the unified Medical Language System meta-thesaurus which powers up the search capability of PubMed. SNPcurator combines the user input (the disease term in the current version) with relevant genetic search terms to construct the following PubMed query:‘{disease} AND’The first step is to identify genetic research studies from the complete PubMed repository without any limitation on citations counts, publishing journals or a certain time period. The NCBI Eutilies . It accomplishes all necessary NLP tasks easily through its Python API. SpaCy is debatably one of the fastest publicly available parsers . The ID format differs between a newly submitted SNP (e.g.: ss28937569) or reference SNP cluster, refSNP (e.g.: rs28937569), where the latter is assigned to a submitted SNP after the sequence is aligned to its appropriate region , which requires normalization to one single standard format that is easy to capture and extract. Following that, a set of predefined regular expressions are used to capture and recognize the format followed by floating, scientific or exponent number notations. However, since a sentence meaning relies on its semantics and structure, the coupling of P-value to its corresponding SNP is not the same for all sentences. The most common and straight forward way of stating results is by mentioning both SNP and P-value in the same sentence. In this type, the extraction of needed pairs <SNP, P-value> relies on the order as illustrated in the examples shown in P-value to the SNP mentions. Note that a detected P-value can only be coupled with a single SNP mention, and thus not considered when measuring the distance for the next SNP. This allows an accurate coupling when multiple values are involved such as in examples . The same process also applies to the extraction of the odds ratio (OR) values reported in abstract text. In general, OR is a measure of the effect size in case-control study and in genomic-studies specifically, it denotes the probability of having the disease n individuals with and without certain genotypes of SNPs.To relate a reported value(s) to mentioned SNPs, the system first highlights one or more ‘result sentence(s)’ from the abstract. A ‘result sentence’ is identified as any phrase with . The spaCy parser is then invoked to search for all numeric modifier (NUMMOD) dependencies found in the abstract text. The keyword sets are first compared against the head token of each candidate modifier and in the case of a no match; they are then compared against a list of two neighboring words of the candidate. Examples of control and patient group sizes extracted from evidence sentences are demonstrated in The sample size included in the study is another key parameter when confirming the association with statistical confidence. For that purpose, we created two sets of keywords that are commonly used to describe both the patient cohort (‘PatientKeywordSet’) and the control group (‘ControlKeywordSet’). The ‘PatientKeywordSet’ consists of words such as  and the ‘ControlKeywordSet’ includes words like ents attribute of the processed input abstract lists all entity objects found. An entity object is a combination of text, category and word position within text. We are particularly interested in the NORP entity which extracts nationalities, religious or political groups efficiently (where the latter two are irrelevant in this context). To limit the number of false positives, we also set the minimum word length for a nationality. If more than one nationality/ethnicity are found, the most frequent nationality mentioned would be reported as shown in Because genetic variants often have markedly different frequencies across populations, the system is also able to extract the ethnicity and nationality of patients through the spaCy name entity recognition module. The http://flask.pocoo.org/). Flask is a micro-framework for Python based on Werkzeug, the WSGI utility library and Jinja temple engine. It provides simple and flexible routing to build python-based web applications and also allows the integration of other python scripts. The disease query is passed from the front-end; the server initiates and displays the output from the text-mining module script. All the extracted information is displayed in a tabular format, where each row includes a single SNP item. The last column allows the user to check the result sentence from which the <SNP, P-value> tuple was extracted. The user is able to sort the results according to the P-value, OR value, group sizes, date of publication or simply alphabetical/numerical order of the PubMed or SNPs Ids. For example, by ranking results according to the ascending P-values, researchers can easily view the SNPs with the strongest association values that attained the genome-wide significance level. The web-tool is deployed on a server connected to the internet and can be accessed at http://snpcurator.science.uu.nl/, SNPcurator web service is implemented in Python and based on the Flask web development library  and it is the first cause of cancer death. Although obesity is not in itself a direct cause of death, overweight is a major risk factor of several diseases leading to death. However, it is also considered one of the top preventable diseases. Therefore, by identifying individuals with increased risk of obesity, an early treatment plan would help to avoid mortal consequences .P-values, SNPcurator shows evidence for SNPs  and SNP (rs11127958) for the lung cancer and the obesity queries, respectively. Even though the GWAS catalog curation constraints might be the reason why these records are not listed in the catalog, we were still able to confirm these associations through other resources . The corpus was constructed by querying the GoPubMed (http://www.gopubmed.org/) with 20 popular SNPs fetched from SNPedia. The original 20 SNPs names were used as seeds for the abstract collection process that resulted in 360 abstracts with 875 distinct SNPs. The novelty of the SNPPhenA corpus relies in ranking the associations by classifying them into three classes: positive, negative and neutral. The associations were manually annotated by two experts in the genetic fields and in case of any contradictory results; the verdict of a third annotator was taken into consideration. Moreover, a confidence level of positive associations was manually extracted based on the strength of the linguistic correlation between SNPs and disease mentions in the abstract, they were categorized into weak, moderate and strong. More details on the annotation process and the corpus statistics can be found at  pairs in terms of precision, recall and F-measure. The model achieved 81, 86 and 83%, respectively for each metric. What mostly affect the system sensitivity is the false negative (FN) cases that represents missed associations that were not detected by our system. Missed associations occurred due to failing to detect the P-value correctly or failing to link the correct P-value to the correct SNP. Failing to detect the correct P-value happens when values are reported in ranges instead of a single value. Another limitation of the system that it can only extract associations in the same sentence, i.e. both SNP and P-value are mentioned in the same sentence. It also assumes that one P-value applies to multiple SNPs if there is a single P-value mention with multiple SNPs mentioned in the sentence which in some cases results in False positives.On the other hand, failing to couple SNPs to their corresponding P-values contributes to a larger portion of (FN) and aslo to FP. To properly evaluate our model, only a subset of the corpus SNPPhenA_mod was used. All records of the original corpus with no p-values/OR values reported in the abstract text were excluded and not considered when building the new corpus. The modified corpus, SNPPhenA_mod, consists of 120 abstracts with 166 key sentences and a total of 331 SNP-Phenotype association candidates with 160 distinct SNP identifiers. The new corpus was constructed following the manual annotation of associations by a biological expert, itis available for download in XML format from the about page. We evaluated the performance of extracting both  reported in abstracts; moreover it extracts population-related information such as the cohort sizes and ethnicity. To illustrate the tool’s usability, we compared the results of two sample queries as opposed to the manually curated database. The system was able to report a comparable number of associations and also recreate a number of existing associations found in GWAS catalog. The system was also able to identify new associations not found in the catalog that might be interesting for experts to further investigate. Furthermore, to evaluate its usefulness and ease of use in daily research practice, a group of experts used the tool to search for any disease of interest; their opinions were very encouraging and confirmed the potential for SNPcurator. The tool was proven scalable and robust by applying it on the whole PubMed repository, and increasing the Faded articles from 1000 to 3000 per query. A main limitation was the analysis of abstract text only, we believe more accurate data would be extracted from full-text articles.In this article, we presented SNPcurator, a text-mining web-tool for automating the curation process of SNPs-trait information from GWASs. The system efficiently extracts associations and matches them with their statical significance parameters (We acknowledge the fact that text-mining systems would never step up to the level of sophistication of researchers when reading and analyzing an article and hence would never fully replace human curation. However, it is also almost impossible to keep up with the fast publication rates of scientific research today. The speed and satisfying results of SNPcurator may be very beneficial to accelerate that process.Results have shown a difference between the amount of associations reported in abstracts and those reported in the article text itself. In the future, we aim to expand the text-mining scope to full-length articles and also any extra data provided by authors to further improve the system’s performance. This would allow to extract more information regarding associations and also detailed cohort descriptions. Another potential add-on to the system would be a disease–SNP relation extraction module that allows an inverse query search by SNP id or even simply a set of PubMed articles ids. The system would also benefit from a general scoring scheme or a filter to rank the results not only based on the statistical findings but should incorporate as well the number of citations and the impact factors of the publications to indicate the credibility of the reported associations.Conflict of interest. None declared.


SNP genotyping platform ‘Axiom®SNPCicer Array’ has been designed, developed and validated. Screening of whole‐genome resequencing data from 429 chickpea lines identified 4.9 million SNPs, from which a subset of 70 463 high‐quality nonredundant SNPs was selected using different stringent filter criteria. This was further narrowed down to 61 174 SNPs based on p‐convert score ≥0.3, of which 50 590 SNPs could be tiled on array. Among these tiled SNPs, a total of 11 245 SNPs (22.23%) were from the coding regions of 3673 different genes. The developed Axiom®SNPCicer Array was used for genotyping two recombinant inbred line populations, namely ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261). Genotyping data reflected high success and polymorphic rate, with 15 140  and 20 018  polymorphic SNPs. High‐density genetic maps comprising 13 679 SNPs spanning 1033.67 cM and 7769 SNPs spanning 1076.35 cM were developed for ICCRIL03 and ICCRIL04 populations, respectively. QTL analysis using multilocation, multiseason phenotyping data on these RILs identified 70 (ICCRIL03) and 120 (ICCRIL04) main‐effect QTLs on genetic map. Higher precision and potential of this array is expected to advance chickpea genetics and breeding applications.To accelerate genomics research and molecular breeding applications in chickpea, a high‐throughput  Cicer arietinum L., 2n = 16) is the second most important grain legume after common bean and third important pulse grown on low‐input marginal lands and known to play a key role in food and nutritional security globally  and high‐throughput genotyping technologies have enabled the use of these technologies at lower cost and offered opportunities to deliver high‐throughput data to capture millions of variations in genome level. With the availability of draft genome sequence  followed by 11 245 SNPs (22.23%) from coding regions and 7688 SNPs (15.20%) from intronic regions , nonsynonymous coding , stop gained (23), synonymous stop (14), stop lost (10), synonymous start (8), splice site donor (8), splice site acceptor (7) and start lost (6).Selected 50 590 SNPs cover all the eight pseudomolecules of chickpea and provide a good representation of whole chickpea genome Figure a,b, with31 653; 6.57% foll Functional annotations of SNPs resulted in detailed classification of involvement of SNP‐carrying genes in (i) biological process, (ii) molecular function and (iii) cellular component Figure c. Under ®CicerSNP Array. ICCRIL03 consisting of 245 lines was genotyped with the developed Axiom®CicerSNP Array. Three samples were removed from the analysis due to below‐threshold standard dish quality control check (DQC < 0.82). Seven additional samples were removed due to a lower call rate (<97%). An initial performance validation of the Axiom®CicerSNP Array followed the Axiom best practices genotyping workflow. SNPs were classified as described in ‘Methods’ section  covering 1033.67 cM   using QTL IciMapping software. In the case of ICCRIL03, a total of 70 QTLs were identified that included 26 QTLs for phenological traits, 11 QTLs for morphological traits, 21 QTLs for yield‐related traits, 6 QTLs for drought indices, 5 QTLs for root traits and 1 QTL for carbon isotope discrimination (δC) Table . HoweverC) Table . Of the In the case of ICCRIL03, three QTL clusters were observed. Cluster one located on CaLG04 represented QTLs for 100‐seed weight , days to 50% flowering (DF), PHT, pods per plant (POD), while other two clusters were located on CaLG08 responsible for DF, days to maturity (DM), harvest index , PHT and DF, DM, PHT traits, respectively. Similarly in the ICCRIL04 population, a total of nine QTL clusters were identified on CaLG01 , CaLG02 (two clusters: a) HI, PS, yield ; (b) biomass , drought tolerance index (DTI), PS, YLD), CaLG03 , CaLG05 (one cluster: DF and seeds per pod (SPD)) and CaLG08 (three clusters: a) DF, DM, HI, PHT, YLD; b) DF, DM, HI, PHT; c) 100‐SDW, DF, DM, PHT, POD).On the basis of location/year, QTLs were further categorized in two categories: (i) QTL for a trait appearing in more than one location was considered as ‘stable’ QTL and (ii) QTL appearing for >1 year or season was considered ‘consistent’ QTL Tables  and 5. P3), root dry weight , rooting depth , root surface area , root volume  and root dry weight/total plant dry weight ratio ] were analysed and QTLs for five traits one each for RLD, RSA, RV, RTR and RDp in ICCRIL03 with PVE ranging from 6.55% (RSA) to 66.49% (RV) were identified , PBS (3 QTLs) and SBS (1 QTL). Overall, PVE ranged from 6.12% (PHT) to 13.51% (PHT) Table . In all,In the ICCRIL04 population, 41 QTLs for morphological traits were identified, of which 17 QTLs were for PHT, 11 QTLs for SDW, 9 QTLs for PS and 4 QTLs for PWD. PVE ranged from 5.49% (SDW) to 17.65% (PHT) among QTLs for morphological traits Table . In totaviz. DF (13 QTLs) and DM (13 QTLs), with PVE ranging from 4.8% (DF) to 34.82% (DF)  Table . Of totaIn ICCRIL04 population, a total of 21 QTLs were observed, of which 10 for DF and 11 for DM with PVE ranging from 5.59% (DF) to 39.32% (DF). A total of 12 robust QTLs were identified, six for each DF and DM Table . Three Qviz. 3 QTLs for 100‐SDW, 3 QTLs for POD, 1 QTL for SPD, 2 QTLs for BM, 8 QTLs for HI and 4 QTLs for YLD in ICCRIL03 population  to 35.88% (HI) Table . There w13C) as a surrogacy trait for transpiration efficiency (TE), only single QTL was identified in ICCRIL03 with PVE of 5.46%  with PVE ranging from 5.58% (DSI) to 8.13% (DTI) Table . However6% Table , while iet al., et al., Molecular markers have been widely used for genetic diversity assessment, evolutionary and mapping studies .In recent past, NGS‐based technologies have been effectively used for genome sequencing and resequencing, enabling the identification of millions of SNP markers in chickpea  were identified in comparison with the previous study , common QTLs were identified for four root traits . However, it was interesting to note that except for RLD, QTLs for these traits were identified on different linkage groups in the RIL populations. Additionally, QTLs for RV and RDW were found only in ICCRIL03 and ICCRIL04, respectively. The possible reason behind this contrasting identification of QTLs in two RIL populations could be either the presence of several small‐effect QTLs or break of robust QTLs into many small‐effect QTLs due to higher number of markers in the current study.et al., ®CicerSNP Array, while working on different mapping populations comparison of genetic with physical positions of the SNP can be used for further validation and improvement of assembled chickpea genome. Some studies in maize and apple have clearly shown the possibility of improvement of genome assembly using large number of markers by applying array‐based genotyping for high‐resolution genetic mapping  and ICC 1882 (a drought‐sensitive genotype), and ICCRIL04 generated by crossing ICC 8261 (a drought‐tolerant genotype) and ICC 283 (a drought‐sensitive genotype), were used in this study.et al., Genomic DNA from parental genotypes and RILs were isolated using high‐throughput mini‐DNA extraction . These datasets have been described in earlier studies  AxiomGTv1 algorithm to ensure a high‐quality final array. Probability of SNP conversion represented by p‐convert value was predicted using random forest model for both forward and reverse probes of each SNP. The model takes into account factors such as sequence of the probe, binding energy and expected degree of nonspecific hybridization to multiple genomic regions. High p‐convert values of probes reflect high probability to convert on the SNP array. In addition, few SNPs selected as part of earlier studies such as results of skim sequencing . SNPs from earlier studies were filtered using ‘in‐house’ multiple sequence alignments scripts and SNPs with no other SNP in the flanking 35 bp on both sides were selected. These selected SNPs were then subjected to seven different kinds of filters, namely (i) no SSR in flanking region, (ii) no Indel in flanking region, (iii) only biallelic nature SNPs, (iv) minor allele frequency of 0.05, (v) GC% to be 40%–70%, (vi) no denaturing code permitted in the region and (vii) SNP quality score ≥30 to select best SNPs for the array. High‐quality SNPs resulted from these assorted criteria were further subjected to ®CicerSNP Array, high‐quality DNA extracted from fresh leaves of both the populations, ICCRIL03 with 245 lines and ICCRIL04 with 230 lines along with parental genotypes, were used for genotyping. In brief, high‐quality total genomic DNA (100 ng) was randomly fragmented (25–125 bp) and fragmented DNA was purified before hybridizing with Axiom®CicerSNP Array. Under stringent conditions, nonspecific random ligations bound to targets were washed off. In order to identify multicolour ligation event at the array surface which points towards polymorphic nucleotide, array was stained, imaged and processed using GeneTitan® Multi‐Channel (MC) instrument to generate the data.In order to utilize the developed Axiom.CEL files’ with Axiom Analysis Suite (1.1.0.616). Axiom best practice workflow was followed in order to get high‐quality genotyping results . These different classes include MonoHighResolution (SNPs with genotyping data with QC pass but monomorphic across the genotypes studied), PolyHighResolution , NoMinorHom , Off‐Target Variant (data with low‐intensity cluster resulted from mismatches between the probe and the sequences for that group of genotypes) and Other (SNP with genotyping data with no clear cluster pattern and could not be assigned any of the other classes). PolyHighResolution SNPs were further filtered out for reproducibility and variance. In addition, markers with complex genotypes causing cluster splits are further filtered into additional classes using ‘Variance’ filters.Allele calling was performed using generated ‘is Suite .1.0.616.et al., In order to identify putative function of SNP‐containing genes, similarity search with a cut‐off value of ≤1E‐5 was performed against existing NR protein sequences in the public database. In order to determine the involvement in particular biological function, these SNP‐containing genes were annotated based on the Gene Ontology (GO) terms associated with the blast results using Blast2GO software (Conesa P < 0.01) were removed from further analysis. The genetic map was constructed from the selected SNPs using R/qtl program  population.Click here for additional data file.Table S2 Linkage group wise SNP data summary in ICCRIL04 (ICC 283 × ICC 8261) population.Click here for additional data file.Table S3 Details on QTLs identified for the 17 traits and two drought indices in ICC 4958 × ICC 1882 population.Click here for additional data file.Table S4 Details on QTLs identified for the 17 traits and one drought indices in the ICC 283 × ICC 8261 population.Click here for additional data file.


Cassava is one of the most important food security crops in tropical countries, and a competitive resource for the starch, food, feed and ethanol industries. However, genomics research in this crop is much less developed compared to other economically important crops such as rice or maize. The International Center for Tropical Agriculture (CIAT) maintains the largest cassava germplasm collection in the world. Unfortunately, the genetic potential of this diversity for breeding programs remains underexploited due to the difficulties in phenotypic screening and lack of deep genomic information about the different accessions. A chromosome-level assembly of the cassava reference genome was released this year and only a handful of studies have been made, mainly to find quantitative trait loci (QTL) on breeding populations with limited variability. This work presents the results of pooled targeted resequencing of more than 1500 cassava accessions from the CIAT germplasm collection to obtain a dataset of more than 2000 variants within genes related to starch functional properties and herbicide tolerance. Results of twelve bioinformatic pipelines for variant detection in pooled samples were compared to ensure the quality of the variant calling process. Predictions of functional impact were performed using two separate methods to prioritize interesting variation for genotyping and cultivar selection. Targeted resequencing, either by pooled samples or by similar approaches such as Ecotilling or capture, emerges as a cost effective alternative to whole genome sequencing to identify interesting alleles of genes related to relevant traits within large germplasm collections. GS synthesizes glutamine and is very important in the regulation of the nitrogen metabolism Resistance to two types of herbicides, inhibiting amino acid biosynthesis, has been commercially exploited in different crops and was targeted in this study. The first group of herbicides , interact with the enzymes Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) Manihot species (more than 6000 accessions). The in vitro collection at CIAT was initiated in 1978 soon after the technology for slow growth in vitro became available CIAT holds in trust the largest global germplasm collection of cassava and other In recent years, the development of high throughput sequencing technologies led to major progress in the understanding of genomic variation in plants, increasing the number of sequenced genomes In this study, we performed pooled targeted resequencing of DNA from 1667 cassava accessions to detect rare SNPs in specific genes associated with the starch biosynthesis pathway and with herbicide resistance. Selected accessions represent about one fourth of the entire collection and include landraces from the most important regions of cassava production in Latin America. We combined the results of 7 variant calling tools applied to aligned reads obtained with two different algorithms to develop a dataset of more than 2000 SNPs within the genes of interest. These SNPs can be prioritized and validated for allele mining and efficient identification of mutated genes in accessions within the cassava germplasm collection.22.1DNA was extracted from a total of 1728 accessions from the germplasm collection (Supplementary Table 1). In general, DNA quality was good, with 70% of the samples showing clear shaped bands without significant smearing (Supplementary figure 1). Only 61 samples were discarded due to low DNA concentration. For pooled resequencing two possible methods to normalize DNA concentration across samples were evaluated: the use of paramagnetic beads and visual concentration determination using agarose gels see . Owing tAmplicon products for each pool were sent to the high throughput sequencing Illumina MiSeq instrument available at the Plant Breeding and Genetics Laboratory from the International Atomic Energy Agency (IAEA) in Seibersdorf, Austria. After one 2 × 300 paired-end sequencing run, around 2.5 million fragments were obtained for each pool. Assuming that these fragments are evenly distributed across the targeted regions, this raw sequencing production represents a expected read depth of around 20,000 × per targeted base pair within each pool. Reads were trimmed to 240 bp for the first read and to 170 bp for the second read to remove low quality ends. Alignment of the trimmed reads to the reference genome yielded an overall alignment rate of 97%, with 89% of the fragments aligning to unique locations and with the expected distance and orientation a. Even r2.2The number of fragments assigned to each region is tightly related to the total read depth available within each particular locus to assess the presence of non-reference alleles, call variation, and estimate relative allele frequencies based the number of reads supporting each allele. Theoretically, if 10,000 fragments are assigned to one region within one pool, the minor allele of a biallelic variant with a frequency of 0.01 within the samples included in the pool should be observed in about 100 reads. Because about 200 samples were included in each pool, heterozygous variants present in only one sample would have a minor allele frequency (MAF) of 1/400 = 0.0025 within one pool. Although in this experiment some of these variants would have enough read support be detected, it becomes increasingly difficult to separate the support of true alleles with low frequency from sequencing errors.To identify sites with evidence of variation within the pools, we combined the results of 12 previously published bioinformatic pipelines designed to discover single nucleotide polymorphisms (SNPs) and in some cases small indels. The pipelines are the combination of 2 read alignment tools, Bowtie2 Merging the variants predicted by the different pipelines, a raw dataset of 7925 variants was obtained, including 7348 biallelic SNPs, 258 biallelic indels and 319 multiallelic variants. Reads supporting each allele of each variant within each pool were counted following the genotyping step of the NGSEP pipeline and allele frequencies were estimated from these counts. About 70% of the raw variants are located within the targeted regions. At first sight, this percentage looks inconsistent with the capture success rate of 91% reported above. The explanation for this outcome is that variants outside targeted regions are called from the few reads falling away from targeted regions and then the total read depth of those variants is much lower than that of the variants within the targeted regions (Supplementary figure 3). The raw variants were filtered by minimum read depth, number of pools in which the variant is observed, and minimum alternative allele frequency. To differentiate true rare SNPs from sequencing errors, the number of errors for each raw SNP was estimated as the average between the third and the fourth smallest allele read depth. Then, the ratio between the read depth of the allele with the second count and the estimated number of sequencing errors was calculated and the SNP was filtered out if this ratio was less than 5. This filtering procedure yielded a curated dataset of 2614 SNPs (Supplementary Table 4). Estimated allele frequencies for curated SNPs were adjusted taking into account read counts of the two predicted alleles. Contrasting the raw calls obtained using each tool during the discovery step with this filtered dataset, we found that 80% of the SNPs in the final set were discovered only by VipR and only 46 SNPs were reported by tools different than VipR. The filters reduced the number of SNPs called by each method to about half in the case of vipR and SNVer, and up to 1 over 10 in the case of Samtools. Samtools only reported 108 of the filtered SNPs with only one SNP not shared by other tools. SNVer and NGSEP were the second and third tools reporting more SNPs within this dataset with 398 and 330 SNPs respectively. The SNPs contributed by the same discovery tool using different read alignment methods were compared to assess the consistency of each method relative to the input alignments b. AlthouIn absence of a gold-standard to perform a formal quality assessment of the variants predicted by different pipelines, we also calculated the intersections between SNP discovery tools, excluding vipR c and d. We also investigated the consistency of allele frequency estimations between pools, taking into account that the samples were pooled without information of population structure and hence the allele frequencies of variants should be stable across pools. Looking for further evidence to assess the precision of the SNP calling procedure, we compared the SNPs predicted in this work with the SNPs identified from an analysis of whole genome sequencing (WGS) data from 58 cassava varieties 2.3SSIII and SSIV show a larger SNP density and for SSIV in particular the number of synonymous mutations (3) is much smaller than the number of missense mutations (11). Six of these missense mutations have a predicted MAF larger than 0.1. The number of non-sense mutations reduced to only seven. Interestingly, two of these mutations, which modify the codons 141 and 143 at exon 4 of the gene GWD showed alternative allele frequencies close to 0.5 and to 0.25 respectively over the 8 pools. Read counts indicate that in almost all pools the alternative alleles of both mutations were supported by over 3000 reads and that the number was always 5-fold higher than the number of reads supporting other alternative allele. Three additional mutations with MAFs larger than 0.15 are located close to the end of the SSIII and the AHAS4 genes.Functional annotations of the dataset of filtered SNPs using both NGSEP and SNPeff were performed, obtaining 317 synonymous, 1037 missense and 59 non sense mutations a. At firUnfortunately vipR and SNVer, which were the two software packages implementing models for pooled sequencing data, were not designed to call small indels. Combining results of the other tools, 4 small indels were identified within coding regions of the sequenced genes (Supplementary Table 5). One of these indels, located within the gene SBE was a missense 3 bp deletion, which removes a lysine amino acid. The three remaining indel mutations are all 1 bp deletions located at the AHAS 4 gene located at chromosome 17 c. The th3The recent releases of chromosome-level assemblies for different plants and the continuous reduction in sequencing costs allows research in staple crops such as cassava to enter the post-genomic era in which comprehensive characterization of genomic diversity across complete genebank collections becomes a feasible task The targeted sequencing strategy followed in this experiment indeed revealed a large amount of variants at different allele frequencies within the targeted genes. A comparison with the SNPs identified by whole genome sequencing of 58 African varieties  served as validation of the variants with high Minor Allele Frequency (MAF) but also showed that sequencing a limited number of varieties does not allow identification of a large amount of genetic variation that could be potentially relevant for breeding purposes. The consistency in predictions of allele frequencies observed across the eight pools suggests that the method employed for DNA normalization and the bioinformatic analysis were generally effective and hence they can be used for future pooled sequencing experiments. The main drawback that we could observe using the pooled targeted sequencing approach was a reduction of the regions effectively sequenced by the experiment due to the increased error rates toward the 3′ ends of the reads. Because reads are directly sequenced from PCR products and not randomly sampled within the targeted regions, high error rates at the 3′ end of the reads will accumulate at the central parts of the targeted regions, producing a large amount of false positives. If reads are trimmed to prevent this effect, central parts of some of the targeted regions are lost. In future experiments, amplicon lengths of PCR products should be reduced to take into account the error rate of the sequencing instrument. A second drawback of this approach is that individual genotyping of the variants revealed by the experiment can not be achieved within the experiment. We are currently evaluating different techniques to perform direct genotyping of the most promising SNPs identified in this work.The most commonly used tools for variants discovery  are not designed to detect low frequency variants in pooled samples, because they were designed to perform variants discovery from alignments of reads sequenced from individual samples. Hence, one of the assumptions to improve the genotyping quality in these tools is that the two alleles in heterozygous sites will have even representation in the sample. This is not the normal case for pooled samples because population allele frequencies determine the relative proportion of read counts supporting each allele within variant sites. However, we could only find two additional software tools (VipR and SNVer) that would be feasible to run on current aligned HTS reads and that implemented statistical models to find the low frequency variants that could potentially be extracted from these data. An initial comparison of the variants obtained with these two tools showed that their results were very divergent, with VipR reporting between five and twelve times more variants than SNVer, depending on the read alignment tool a. AlthouThe final outcome of the comparison between pipelines for variants discovery and the filtering process, including the filtering of variants in which the minor allele could not be clearly separated from sequencing errors, is a dataset of 2614 SNPs within the targeted genes (Supplementary Table 4). Despite of the filtering procedure, close to 80% of these variants are still SNPs with low MAF identified only by VipR. Although we could follow a more conservative approach and report only SNPs called by a certain type of intersection between the tools, this would remove most of the rare mutations that are actually interesting for follow up genotyping experiments. For this reason, we decided to retain the union of the SNPs identified by the different tools after performing the filters described above. However, each SNP is reported with functional annotations, intersection with SNPs obtained from WGS data, predicted allele frequencies, raw read counts and pipelines that reported each variant. This allows different researchers to use common excel filters to select the most appropriate variants for different follow up experiments.AHAS genes seem to have larger variability than the genes related to starch production, even after normalization by the covered portion of coding regions. GBSSI is the gene with the lowest variability, probably because it is the main enzyme that catalyzes the reaction to produce amylose. Conversely AHAS4 shows the largest number of variants and also contains three frameshift indels that potentially produce silencing of this paralog. Other interesting variants are the non-sense mutations identified in the single copy GWD gene. If these mutations have a silencing effect, plants carrying these SNPs could accumulate abnormally high levels of starch as shown in previous studies Given the total length of the targeted region, the SNPs identified in this study amount to a density of one SNP for each 26 base pairs. Although we initially found this number surprisingly high, the latest release of the 3000 rice genomes project a-priori could be thought as completely conserved, is also encouraging to try alternative cost-efficient techniques such as multi-dimensional pooled EcoTILLING The SNPs identified in this study can be prioritized based on read evidence and predictions of functional consequences, and then they can be tested in a direct genotyping platform. We are currently exploring different alternatives to perform individual genotyping, not only for validation but also to identify varieties with rare alleles that could exhibit interesting characteristics for the traits of interest that then could be selected as new sources of genetic variability for the cassava breeding program. The publication of the SNPs identified in this experiment is helpful to encourage other groups to perform individual genotyping of these SNPs in their own germplasm collections, accelerating the discovery of varieties with improved phenotypes. Moreover, the genetic variation that we could identify in the CIAT collection, within genes that 44.1DNA was extracted from a total of 1728 accessions from the germplasm collection at CIAT. The DNA was isolated by using 1 g of cassava leaf tissue grounded with liquid nitrogen in 15 mL tubes using the CTAB method. Thereafter, 3 mL of the prewarmed extraction buffer was added (100 mM tris HCl (pH 8), 20 mM EDTA (pH 8), 2 M NaCl, 2% CTAB (w/v), 2% PVP) to each sample and they were mixed. The samples were incubated at 65 °C for 1 h with frequent swirling. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to each sample and mixed gently for 30 min. The samples were centrifuged at 3000 rpm for 30 min at room temperature. Approximately 2 mL of the supernatant was transferred to a new tube. The supernatant was precipitated with 1/1 volume of isopropanol and was incubated for 30 min at 4 °C. The precipitated nucleic acids were collected and washed twice with 70% ethanol. The obtained nucleic acid pellet was air-dried until the ethanol was evaporated and dissolved in 200 uL of TE buffer . The nucleic acid dissolved in TE buffer was treated with ribonuclease A  and incubated at 37 °C for 30 min. The quality of extracted DNA was stained with SYBR safe (Invitrogen) and visualized by agarose gel electrophoresis (1%). The purity of the DNA was estimated by spectrophotometry, which estimates A260/280 and A260/230 ratio. After this, the dried samples were packed to be shipped to the Plant Breeding and Genetics Laboratory in Austria.4.2Once the DNA samples arrived to the Plant Breeding and Genetics Laboratory in Austria for processing and sequencing, were centrifuged and then hydrated by the addition of 100 uL (water). Samples were incubated at room temperature for 10 min followed by a short vortex and an additional 5 min incubation to ensure that DNA was completely in solution. Samples were stored at 4 °C for a minimum of 24 h prior to additional processing.e.g. MagQuantTM) yielded inconsistent concentrations, possibly due to variations of input DNA (data not shown). Therefore a system using 96 well gels and image based quantification was employed To ensure even sequencing coverage of all DNA samples in a pool, methods were evaluated to normalize DNA concentrations. Experiments employing paramagnetic bead-based purification systems , and starch biosynthesis, . Primer3 A total of 121 primer pairs were designed for the exonic regions of genes related to herbicide tolerance (4.4Illumina library preparation was performed using the TruSeq® Nano DNA Library Prep (version 15041110 Rev. D) with minor modification. Briefly, the first normalization and fragmentation steps were not performed and library preparation began with the first bead-based cleanup step. All other steps were followed according to the protocol. Dual indexes were used. Quantification was performed using Qubit fluorometry. Libraries were normalized to 4 nM and pooled together. The concentration of this pool was further checked, adjusted, and the pool denatured and diluted to 17.5 pM according to the Illumina protocol. Samples were sequenced on an Illumina MiSeq using 2 × 300 Paired End version 3 chemistry. Fastqc 4.5Manihot esculenta v6.1 was downloaded from the webpage of Phytozome 11 The reference genome 4.6Seven variant callers were combined with the two read alignment tools to obtain twelve different pipelines. The procedure for each pipeline is briefly described below.4.6.1Freebayes v1.0.2-33-gdbb6160 4.6.2To run GATK 3.5-0-g36282e4 4.6.3The NGSEP-3.0.1 4.6.4The variant calling was performed according to the documentation (version 1.3.1) 4.6.5SNVer-0.5.3 4.6.6To run VarScan v2.3.9 4.6.7This program was executed according to the documentation available (version 0.0.16) 4.7At the end 12 VCF files were obtained as a result of the combination of alignment files made with bowtie2 and BWA and the 7 variant callers. With these 12 VCF files the NGSEP pipeline was used to do the genotyping, first merging the variants present in all the VCF files, and then running the genotyping process with default parameters, except for the maximum number of alignments allowed to start at the same reference site, which was set to 0. This was done with the BAM files for each read alignment tool, generating two final VCF files.The functional annotation was performed using NGSEP and SNPeff


Paired-end reads were collected on an Illumina 3000HT, then aligned to the most recent zebrafish reference genome (GRCz10). These data were used to compare observed population genetic variation across species , then across lines within zebrafish. We found more single nucleotide polymorphisms (SNPs) in T5D than have been reported in SNP databases for any of the WIK, TU, TL, or AB lines. We theorize that some subset of the novel SNPs may be shared with other zebrafish lines but have not been identified in other studies due to the limitations of capturing population diversity in pooled sequencing strategies. We establish T5D as a model that is representative of diversity levels within laboratory zebrafish lines and demonstrate that experimental design and analysis can exert major effects when characterizing genetic diversity in heterogeneous populations.Toxicological and pharmacological researchers have seized upon the many benefits of zebrafish, including the short generation time, well-characterized development, and early maturation as clear embryos. A major difference from many model organisms is that standard husbandry practices in zebrafish are designed to maintain population diversity. While this diversity is attractive for translational applications in human and ecological health, it raises critical questions on how interindividual genetic variation might contribute to chemical exposure or disease susceptibility differences. Findings from pooled samples of zebrafish support this supposition of diversity yet cannot directly measure allele frequencies for reference versus alternate alleles. Using the Tanguay lab Tropical 5D zebrafish line (T5D), we performed whole genome sequencing on a large group ( Danio rerio) as a model organism has gained momentum in vertebrate genomics (Lieschke and Currie Use of the zebrafish (Drosophila melanogaster)  panel, the lack of variability within any single inbred strain did not sufficiently model human genetic variability  line as a representative laboratory population of zebrafish. The T5D line is an “outbred” population of heretofore unknown genetic heterogeneity that has been used to screen thousands of chemicals for adverse biological responses   free line . In brief, genomic DNA was extracted (Zymo Quick-DNA 96-Kit Cat # D3011) from 276 individual larvae exposed to 0.6 µM Abamectin at 120-h post fertilization. The authors note that Abamectin is non-genotoxic . For these samples, 350 ng of DNA was used in the library preparation. Prior to library prep, the quality and quantity were verified using a fluorometric plate reader and Bioanalyzer. Samples were sheared to ~ 320 bp, and 100 ng was used in the WaferGen robotic DNA library prep. After the library prep, each sample was quantified to verify similar input for sequencing. The samples were sequenced on an Illumina HiSeq 3000 with 12 samples per lane (~ 5× coverage) and 150 bp paired-end sequencing.The sequencing data are described in detail in , where there was sufficient evidence  of a non-reference base for at least one individual. GATK  files of SNP and indel variation from four other zebrafish lines , compiled through integration of data from three previous studies . Variants located in these non-complex regions of the genome were removed from the final T5D comparison dataset resulting in 10,301,547 SNPs and 2,375,455 indels. To ensure consistency between datasets, we performed the same masking procedure on the AB, TU, TL, and WIK datasets even though masking had been previously performed. All comparisons with these lines were based on the following approximate counts .Additionally, the CVF files had masked variants in non-complex regions of the genome. To filter T5D variants accordingly, the repeat masked annotation of Zv9 was downloaded from A VCF file for NHGRI-1 .A T5D-specific reference was created. SNP and indel VCF files based on the GATK best practices recommendations were used. The indel file was further filtered to remove known repeats in the GRCz10 reference build. This minimized differences called based on microsatellites and other variable number tandem repeats (VNTRs). A bed file of all known The original and new genomes were split by chromosome for comparison using the nucmer package from the software MUMmer. Nucmer was run with the—mum option. The resulting delta files were filtered for 1-to-1 alignments allowing for rearrangements, and the filtered delta files translated to coordinates to be used in MapView for plotting. The filtered delta files were also run through dnadiff.The zebrafish genome 1.5 Gb) is roughly half the size of the human (3.3 Gb) or mouse (2.8 Gb) genome. To date, the total number of discovered variants in the zebrafish genome is less than half the number found in human or mouse genomes; consequently, validation is more sparse. The allele frequency distribution of “common” human variants indicates that the majority of common variants are infrequent across the overall human population [minor allele frequency (MAF) < 0.1]  included non-reference allele frequencies from 0.1 to 99.8%. We posit that the 10.3–20.1 M SNPs and 2.8–5.6 M indels discovered in T5D are accurate bounds for an estimate of variability in this zebrafish line. With more individuals and higher coverage, we would expect to find even more rare variants segregating in the population. This would be consistent with the continued rare variant discovery in human populations noted in the previous section.With the exception of chromosome 4, the number of variants discovered per chromosome was proportional to chromosome length  genome sequence per strain, which is still more than in any ethnically defined human population from the 1000 Genomes  at 6.9 M SNP sites and 1.8 M indel sites (3.7 M SNP sites and 0.84 M indel sites in non-masked genomic regions). This is more than have been identified in individual human genomes. For example, in Caucasians an average of 3.3 M SNPs and 0.49 M indels with non-reference alleles were identified per individual  yet missed in the sampled subsets.For the previously discovered variants in AB, TU, TL, and WIK, SNPs in TU followed a slightly different read frequency distribution, with fewer fixed SNPs. This can be explained in part by the heavy reliance of the reference genome sequence on TU zebrafish. Additionally, AB and TU had even fewer low-frequency SNPs, which can be explained by the lower average read depth per SNP site (median of 8 for AB and 9 for TU compared with 16 for TL and 13 for WIK).In order to assess the similarity of T5D variation to a hybrid population that has previously employed an individual sequencing approach, SNP sites were compared to NHGRI-1 SNP sites. The NHGRI-1 line was derived from one mating pair of TAB-5 (a TU and AB cross), where the founding male was previously sequenced at 52× coverage and the female at 47× . This may be partially explained by the lower coverage per individual in our design, wherein we sacrificed sequencing depth per individual in order to include a larger sample and better estimate genotype frequencies for rare variants. These rare variants would not be captured without a reasonably large sample of individuals.In order to assess whether sequencing design could be a major driver behind observed SNP differences between lines, we used a downsampling strategy to approximate published designs used for other lines. We simulated a pool of 20 T5D individuals with average coverage of 20× across the genome by using a subset of the sequencing reads and analyzing them as one pooled sample. Even before applying filters, 49.8% as many variants were detected in this pooled sample compared to the whole dataset. After the simulated analysis, median read depth per variant site for T5D was 14 (within the range of 8–16 mentioned previously for the other 4 lines).T5D variant counts and proportions of non-reference reads moved closer to those observed in other lines Fig. . Low-freWe used new data from a genome-wide sequencing project to compare and characterize observed population genetic variation across species . While more variants have been discovered in the human and mouse genomes, the smaller zebrafish genome is on par with—or in some cases may even exceed—genetic variability observed between individuals in those species. This diversity is attractive for translational applications in human and ecological health, where natural genetic variability could manifest as susceptibility differences to chemicals, drugs, environmental change, or other stressors. Though there are fewer zebrafish disease models compared to other species Fig. c, the nuVariant discovery in the T5D wild-type zebrafish has confirmed the line’s status as a heterogenous population. Considerably more SNPs and indels were discovered through individual whole genome sequencing of a large T5D sample than in other zebrafish studies, even exceeding the current build of dbSNP. Pooled sequencing data fundamentally affected the character of genetic variation previously detectable in outbred zebrafish lines, versus the individual-level sequencing data collected for T5D. In addition to discovering more variants, the design allowed us to estimate allele frequencies for a population more accurately than previously possible due to bias when estimating based on read frequencies in a pool . Risk assessment can be improved significantly with actual knowledge of subgroup and chemical-specific genetic variability  . Thus, inclusion of knowledge regarding constitutive genetic diversity will benefit all translational applications of the zebrafish model, from the mechanistic to the ecological to the clinical.Additionally, population genetic information can be used to determine variants  associated with differential chemical responses (Balik-Meisner et al.,


The nonlinear saturable and polarization-induced absorption of ReS2 are investigated at near-infrared communication band beyond its bandgap. It is found that the ReS2-covered D-shaped fiber (RDF) displays the remarkable polarization-induced absorption, which indicates the different responses for transverse electric (TE) and transverse magnetic (TM) polarizations relative to ReS2 plane. Nonlinear saturable absorption of RDF exhibits the similar saturable fluence of several tens of μJ/cm2 and modulation depth of about 1% for ultrafast pulses with two orthogonal polarizations. RDF is utilized as a saturable absorber to achieve self-started mode-locking operation in an Er-doped fiber laser. The results broaden the operation wavelength of ReS2 from visible light to around 1550 nm, and numerous applications may benefit from the anisotropic and nonlinear absorption characteristics of ReS2, such as in-line optical polarizers, high-power pulsed lasers, and optical communication system.Monolayer of transition metal dichalcogenides (TMDs), with lamellar structure as that of graphene, has attracted significant attentions in optoelectronics and photonics. Here, we focus on the optical absorption response of a new member TMDs, rhenium disulphide (ReS The nonlinear saturable and polarization-induced absorptions of ReS2 are demonstrated at infrared communication wavelength in our work. ReS2 is transferred onto a side-polished surface of optical fiber, where light propagates parallel to the ReS2 layer in one direction. The ReS2-covered D-shaped fiber (RDF) displays the remarkable polarization-induced absorption, while the nonlinear saturable absorption possesses the features that are independent of polarization. The performances of RDF are further verified in an erbium-doped fiber (EDF) laser in which ultrafast pulses are generated at 1564 nm. This work demonstrates the optical performance of ReS2 at near-infrared region beyond its bandgap.Here, we study a new member of the TMDs family, rhenium disulphide  grown on sapphire substrate, which is cut into strip samples with the width of ~1 mm and the length of ~5 mm. The ReS2 sample is transferred from the substrate onto the D-shaped fiber via the wet transfer method like that of graphene22 coated with polymethylmethacrylate (PMMA) is cut into strip samples. Then ReS2 strips are separated from the substrate in the etchant that is removed by rinsing several times in the deionized (DI) water. Finally, ReS2 strips are transferred onto the side-polished area in DI water. The detailed fabrication procedure of the RDF SA is provided in the experimental section. The schematic diagram of RDF SA is shown in 2, which can be regarded as the zoom-in image of 2, which illustrates a distorted 1 T structure. Re atoms form zigzag chains along the b-axis shown in 29β in x-y plane is defined as the angle between the polarization direction of light and the ReS2 layer. And x-y plane is perpendicular to the light transmission direction. For β = 0°, the light polarization direction is parallel to ReS2 plane. In this case, it is defined as transverse electric (TE) wave. For β = 90°, the light polarization is perpendicular to ReS2 plane, which is defined as transverse magnetic (TM) wave.D-shaped fiber is produced by polishing a side of optical fiber into the fiber core, as shown in −1 correspond to the in-plane vibration modes (Eg-like) of Re atom and the low frequency peaks around 134 and 140 cm−1 correspond to the out-of-plane vibration modes (Ag-like)292 is electronically and vibrationally decoupled2 is a kind of in-plane anisotropic 2D material, the line widths and the peak intensities of all the Raman peaks vary with the polarization of probe laser−1) and Ag-like (150.7 cm−1) is ~17.3 cm−1, which indicates the monolayer structure2 is not uniform and is composed of monolayer and multilayer structures. However, the similar Raman results like that in 2 film is monolayer. 2 film on the polished surface of the D-shaped optical fiber. The areas on the both sides of D-shaped fiber are the transition of the film from polished surface to substrate. The edge of film can be observed in 2 and a small amount of evanescent field leak out with ReS2 film.The samples are characterized by Raman spectrometry with the excitation laser wavelength of 633 nm. The non-resonance Raman scattering measurement result is shown in 2 is performed with the experimental setup shown in 2 in a polar coordinate. It can be obviously observed the ReS2-light interaction behavior with the polarization angles. For the TE and TM polarizations relative to the ReS2 plane, it shows the maximum and minimum absorption, respectively. The polarization-dependent performance should be attributed to the presence of ReS2. However, no obvious polarization-induced loss can be observed for D-shaped fiber without ReS2 in our experiments and in previous reports before the graphene transference382, the loss of the D-shaped optical fiber is ~0.5 dB, while the losses for TE and TM polarizations are 4.4 and 2.6 dB with ReS2, respectively.The polarization-induced absorption of the D-shaped fiber covered with and without ReSThe nonlinear saturable absorption of RDF SA is measured with ultrafast pulses at different polarization angles. The experiment setup is illustrated in 5α(F) is the intensity-dependent absorption coefficient, and α0, αns and Fsat are the linear limit of saturable absorption, nonsaturable absorption, and saturation fluence, respectively. When α0, αns and Fsat can be obtained. With the lower nonsaturable absorption, the linear limits of the saturable absorption (α0) and the saturation fluence (Fsat) are approximately 1.2% and 62 μJ/cm2, respectively. With the higher nonsaturable absorption, it shows a smaller saturation fluence (~27 μJ/cm2), while the modulation depth (~1%) is equivalent to that with the lower nonsaturable absorption.where 2 is shown in A schematic of the fiber laser mode-locked by ReS2. Under the net negative dispersion, conventional soliton can generate due to the balance between dispersion and nonlinearity of fiber22 profile, a full width at half maximum is ~1.9 ps, and the deconvolution yields the pulse duration of 1.25 ps. TBP is calculated as 0.4, which means that the pulses are slightly chirped. The pulse train shown in In the RDF SA-based Er-doped fiber laser, continuous wave (CW) is achieved at a pump power less than 5 mW, which benefits from the low loss of RDF SA. By increasing the pump power to ~20 mW, self-started mode locking operation is achieved. However, the mode-locking operation is sensitive to the state of PC since the RDF SA introduces a polarization-dependent absorption. In fact, mode locking operation can be self-started at ~8 mW with the appropriate state of PC. At this time, there are multiple pulses circulating in the laser cavity. The hysteresis phenomenon exits in the mode-locked fiber laser22 has a direct bandgap of ~1.8 eV and the bandgap of the bulk MoS2 is indirect with the range of 0.9–1.3 eV6182 has been reported in numerous previous studies292 have the nearly identical band structure with the direct bandgap of ~1.4 eV2 can be used as SA in a broad operation wavelength18623et al. theoretically investigate the atomic defects in monolayer ReS2, and the densities of states of monolayer ReS2 with different atomic defects were calculated2 (~1.4 eV). The results should originate from the atomic defect in ReS2, because the imperfections formation, e.g., point defects and grain boundaries, are unavoidable during the growth and transfer process.Single layer of TMDs with lamellar structures like that of graphene have received much attention because some of them are semiconductors with sizable bandgaps2, D-shaped fiber-based evanescent field interaction scheme provides a platform to investigate the in-plane characteristics, as light propagates parallel to the ReS2 layer in one direction. 2 are different for TE and TM polarizations41In previous works, SAs were generally fabricated by placing the nanomaterials between two fiber ends, which limits the interaction distance15302478382 have been reported to possess the polarization-dependent absorption, ReS2 displays some unique properties. As the monolayer and multilayer ReS2 have the similar direct bandgap, the experimental results are insensitive to the thickness of sample. In contrast, only monolayer graphene and MoS2 have the direct bandgap, and their band structures vary with the thickness12212 shows the similar merits as discussed above. For example, the saturable intensity of monolayer MoS2 is about several tens of MW/cm2, while it increases at least ten times for multilayer MoS262, the characteristics of saturable absorption would be identical. In addition, the saturable absorptions for two orthogonal polarization pulses were rarely measured in the D-shaped fiber scheme. It is significant for the development of saturable absorber based on D-shaped fiber to show that RDF exhibits the similar saturable fluence and modulation depth for ultrafast pulses with two orthogonal polarizations.Although graphene and MoS2 maintains the characteristics that the bandgap does not change much from monolayer to bulk state. As a result, ReS2 with the different thicknesses display the similar optical properties. The influence of the thickness on the results in the experiment is discussed as follows. Firstly, the similar experimental results could be achieved with shorter length of ReS2 film when the thicker ReS2 sample is used, because it possesses the stronger absorption than monolayer ReS2. But the linear loss would also increase due to the larger perturbation to the evanescent field. When the ReS2 with too large thickness is used, the results cannot be achieved. Secondly, the fabrication of perfect monolayer ReS2 require the strict growth condition, such as time, temperature, catalyst et al., while the grown of thin ReS2 film without thickness limitation needs much lower condition. Finally, here the performance of ReS2 around 1550 nm depends on the atomic defect which is related to the growth condition. However, multilayer ReS2 with different growth condition may obtain the distinct results.ReS2 experimentally with assistant of D-shaped fiber. The linear absorption of RDF changes periodically with the polarization angle, which indicates the in-plane different absorptions of ReS2 for TE and TM polarizations. The optical responses to different polarized ultrafast pulses exhibit the similar features, such as the modulation depth and the saturable fluence, although it exhibits the polarization-induced nonsaturable absorption. The nonlinear absorption of RDF is applied to the ultrafast fiber laser as a SA. Self-started mode-locking operation can be always achieved, which is independent of the state of PC. The results confirm the excellent performance of RDF as a saturable absorption device, which has significant potential applications in a wide wavelength range from visible to mid-infrared (~0.35 eV).In conclusion, we investigated the linear and nonlinear optical responses of ReS2 is produced by chemical vapor deposition (CVD) grown on sapphire substrate. The optical fiber is side-polished into the fiber core with the distance from fiber core to the surface of ~2 μm and the polishing length of ~20 mm, which is fixed on a glass substrate with the polished surface upward as shown in 2, polymethylmethacrylate (PMMA) is spin-coated uniformly onto ReS2 and dried for several hours. Then it is cut into strip samples with the width of ~1 mm and the length of ~5 mm. The samples are put into the etchant solution to detach ReS2 from the substrate. The resulted PMMA/ReS2 strips are transferred into deionized (DI) water three times to rinse the etchant and residues. Subsequently, the side-polished fiber is immerged into the DI water where the ReS2 strips are floating. The position and direction of the sample are carefully controlled with a probe. When the strip is placed on the top of the side-polished area, the D-shaped fiber is lifted out of the DI water, and PMMA/ReS2 spontaneously covered on it. The schematic of ReS2-clad microfiber is shown in 2/PMMA film on the polished side of the D-shaped optical fiber.ReSThe polarization-induced absorption is implemented with the setup shown in 2 is shown in 2.A schematic of the fiber laser mode-locked by ReSAn optical spectrum analyzer (Yokogawa AQ-6370), an autocorrelator, a 6-GHz oscilloscope, a radio-frequency (RF) analyzer, and a 3-GHz photodetector are used to measure the laser output performance.How to cite this article: Cui, Y. et al. Nonlinear Saturable and Polarization-induced Absorption of Rhenium Disulfide. Sci. Rep.7, 40080; doi: 10.1038/srep40080 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.