the fastq file for illumina genome analyzer iix ( gaiix ) run accession srr944107 ( single - end reads ) was downloaded from http://www.ebi.ac.uk/ena/data/view/srr944107 , having sourced the accession code via http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse49302 .
briefly , hek293 t cells were suspended in 1% low - melt agarose prior to lysis .
free dna ends ( sites of dsbs ) were ligated to a double - stranded biotinylated adapter oligonucleotide before digestion with the restriction endonuclease sau3ai .
dsb site - containing termini were phase - purified using streptavidin paramagnetic particles , eluted via ecori restriction endonuclease digestion and then subjected to sau3ai site adapter ligation and pcr amplification .
pcr products were ligated to illumina adapters , allowing them to be represented in either orientation .
library fragments of ~  200400  bp ( insert plus adapter and pcr primer sequences ) were band isolated from agarose gels and the purified libraries were sequenced in single - ended fashion using the illumina genome analyzer iix sequencing platform .
we used our custom software image 1 to produce a modified representation of image 2 .
briefly , it filters reads based on the observation of expected arrangements of adapter sequences , with the stringent requirement that both adapters be evident in a given read .
reads exhibiting evidence of ligation artefacts or insufficient evidence of expected adapter sequences were removed .
accepted reads were processed to trim adapter sequences , and those with library inserts greater than or equal to 25 nucleotides in length were retained and transformed to orient the dsb site at the start .
the concatenated sequences of image 3 plus human reference genome build image 4 , represented as image 5 , were indexed using bwa ( version 0.7.5a )   using the command : reads of the transformed fastq file were then mapped to image 7 using bwa , thus : samtools ( version 1.3.1 )   was used to convert from sam file format to bam file format and to sort the resulting bam file with the following command : bedtools ( version 2.17.0 )   was then employed to produce a bed file representing the mapping , including cigar string information and mapping orientation , with the following command : to reduce false positives resulting from mapping artefacts , we filtered out reads that overlapped with encode project   blacklist regions and repeatmasker - derived repetitive regions as follows ( image 14 represents a file created by sorting a concatenation of the hg19 co - ordinate - associated files image 15 and image 16 ) : we then used our custom software image 19 ( available at https://github.com/djpark1974/raft_hotspots_se ) to further filter the data and to count the number of observations of dsbs at co - ordinates in image 20 ( yielding image 21 ) .
since we presented the dsb at the beginning of each read prior to mapping , we can determine the exact location of the dsb at the single nucleotide level for each read .
reads that mapped in either orientation were treated as likely to be erroneous if the cigar string showed evidence of clipping at either terminus .
additionally , we required reads to exhibit mapping qualities ( mqs ) of greater than 40 for them to be included in our dsb site counting . for increased specificity , we removed dsb sites located within 5 base pairs of a sau3ai consensus site ( gatc ) , thus ( image 22was derived via a custom python script ) : to enable detailed downstream analyses , we have supplemented the co - ordinate - dsb - count data with annotation derived from the encode project and blast alignment scores derived from aligning u13369.1 to the human genome . encode annotations are recorded with the identity and proximity of respective encode elements and blast alignment scores provide the highest scoring sequence similarity match for a contiguous sequence spanning a given co - ordinate .
2 illustrates the frequency of dsbs at single nucleotide resolution sites across the hg19 reference human genome .
the fastq file for illumina genome analyzer iix ( gaiix ) run accession srr944107 ( single - end reads ) was downloaded from http://www.ebi.ac.uk/ena/data/view/srr944107 , having sourced the accession code via http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse49302 .
briefly , hek293 t cells were suspended in 1% low - melt agarose prior to lysis .
free dna ends ( sites of dsbs ) were ligated to a double - stranded biotinylated adapter oligonucleotide before digestion with the restriction endonuclease sau3ai .
dsb site - containing termini were phase - purified using streptavidin paramagnetic particles , eluted via ecori restriction endonuclease digestion and then subjected to sau3ai site adapter ligation and pcr amplification .
pcr products were ligated to illumina adapters , allowing them to be represented in either orientation .
library fragments of ~  200400  bp ( insert plus adapter and pcr primer sequences ) were band isolated from agarose gels and the purified libraries were sequenced in single - ended fashion using the illumina genome analyzer iix sequencing platform .
we used our custom software image 1 to produce a modified representation of image 2 .
briefly , it filters reads based on the observation of expected arrangements of adapter sequences , with the stringent requirement that both adapters be evident in a given read .
reads exhibiting evidence of ligation artefacts or insufficient evidence of expected adapter sequences were removed .
accepted reads were processed to trim adapter sequences , and those with library inserts greater than or equal to 25 nucleotides in length were retained and transformed to orient the dsb site at the start .
the concatenated sequences of image 3 plus human reference genome build image 4 , represented as image 5 , were indexed using bwa ( version 0.7.5a )   using the command : reads of the transformed fastq file were then mapped to image 7 using bwa , thus : samtools ( version 1.3.1 )   was used to convert from sam file format to bam file format and to sort the resulting bam file with the following command : bedtools ( version 2.17.0 )   was then employed to produce a bed file representing the mapping , including cigar string information and mapping orientation , with the following command : to reduce false positives resulting from mapping artefacts , we filtered out reads that overlapped with encode project   blacklist regions and repeatmasker - derived repetitive regions as follows ( image 14 represents a file created by sorting a concatenation of the hg19 co - ordinate - associated files image 15 and image 16 ) : we then used our custom software image 19 ( available at https://github.com/djpark1974/raft_hotspots_se ) to further filter the data and to count the number of observations of dsbs at co - ordinates in image 20 ( yielding image 21 ) .
since we presented the dsb at the beginning of each read prior to mapping , we can determine the exact location of the dsb at the single nucleotide level for each read .
reads that mapped in either orientation were treated as likely to be erroneous if the cigar string showed evidence of clipping at either terminus .
additionally , we required reads to exhibit mapping qualities ( mqs ) of greater than 40 for them to be included in our dsb site counting . for increased specificity , we removed dsb sites located within 5 base pairs of a sau3ai consensus site ( gatc ) , thus ( image 22was derived via a custom python script ) : to enable detailed downstream analyses , we have supplemented the co - ordinate - dsb - count data with annotation derived from the encode project and blast alignment scores derived from aligning u13369.1 to the human genome .
encode annotations are recorded with the identity and proximity of respective encode elements and blast alignment scores provide the highest scoring sequence similarity match for a contiguous sequence spanning a given co - ordinate .
2 illustrates the frequency of dsbs at single nucleotide resolution sites across the hg19 reference human genome .
here , we present the relative frequencies of dsbs across the human reference genome for hek293 t cells , at single nucleotide resolution . since dna strand breakage and genomic rearrangements are highly relevant to cancer and other diseases , , , it is probable that our new data will have utility for the development of clinically important diagnostic tests .
the highest ranking dsb regions reported by tchurikov et al .   for the srr944107.fastq dataset predominantly relate to regions that would be likely to present problems to short read mapping software , such as satellite sequences . in an attempt to reduce mapping - related artefacts ,
we have elected to remove regions known to result in low - confidence mapping from our analysis .
top ranking single nucleotide - resolved dsb sites resulting from our analysis relate to regions listed previously as enriched for dsbs , albeit to a lesser extent than reported for numerous low - complexity ( and low confidence ) sequence regions .
the raft protocol from which our data are derived is theoretically enriched for blunt - ended forum domain termini , previously shown to be associated with transcriptional control , , .
they will be biased towards termini that occur within a particular range of genomic distances from a sau3ai restriction endonuclease site .
future protocols that make use of multiple restriction endonucleases for cleavage following the initial ligation step , as alternatives to ( and as well as ) sau3ai , should mitigate this to a large extent .
we have applied high - stringency thresholding on mapping quality as part of our algorithm and discarded library elements that could not be uniquely assigned to a single genomic location with high confidence and , as such , repetitive genomic elements harbouring dsb sites will not be represented .
it should be noted that the data we present relate to human hek293 t cells .
other cell - types will likely exhibit differences in their raft - detectable dsb profiles due to variations in higher - order chromosomal architecture and dna cleavage - inducing enzyme activity .
these differences will be elucidated with the expansion of studies to a range of cell and tissue types .
we have supplemented the profiling of the relative frequency of dsb sites in hek293 t cells with encode - derived annotation , including regional information pertaining to important transcription factor binding sites and other marks of gene regulation , regions of dnasei hypersensitivity and repetitive elements .
further , we provide annotation in the form of sequence similarity scores , derived from blast analysis , for sites that occur in regions with high similarity to human ribosomal dna , since such sequences are known to include hot spots for dsbs and present particular mapping challenges due to their representation at high copy number at multiple sites in the genome
. this information should assist with the selection of suitable targets for diagnostic test design , allowing the user optionally to avoid sites that present excessive mapping difficulties or to focus on regions associated with particular genomic marks , for example .
the refined characterisation of the propensity for particular types of dsbs , such as those identified by the raft procedure , across the human genome will likely allow more efficient assessment of genomic 
. this should be highly relevant to clinical management approaches such as risk stratification for particular types of cancer and treatment response prediction .
as such , the use of these data has the potential to be beneficial to the reduction of disease associated mortality and morbidity .
 constitutional biological processes involve the generation of dna double - strand breaks ( dsbs ) . 
 the production of such breaks and their subsequent resolution are also highly relevant to neurodegenerative diseases and cancer , in which extensive dna fragmentation has been described stephens et al . 
 ( 2011 ) , blondet et al . 
 ( 2001 ) . 
 tchurikov et al . 
 tchurikov et al . 
 ( 2011 , 2013 ) have reported previously that frequent sites of dsbs occur in chromosomal domains involved in the co - ordinated expression of genes . 
 this group report that hot spots of dsbs in human hek293 t cells often coincide with h3k4me3 marks , associated with active transcription kravatsky et al . 
 ( 2015 ) and that frequent sites of dna double - strand breakage are likely to be relevant to cancer genomics tchurikov et al . 
 ( 2013 , 2016 ) . 
 recently , they applied a raft ( rapid amplification of forum termini ) protocol that selects for blunt - ended dsb sites and mapped these to the human genome within defined co - ordinate  
 windows. in this paper , we re - analyse public raft data to derive sites of dsbs at the single - nucleotide level across the built genome for human hek293 t cells ( https://figshare.com/s/35220b2b79eaaaf64ed8 ) . 
 this refined mapping , combined with accessory encode data tracks and ribosomal dna - related sequence annotations , will likely be of value for the design of clinically relevant targeted assays such as those for cancer susceptibility , diagnosis , treatment - matching and prognostication . 