Abstract
BackgroundCharacterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing. Reliance upon short DNA fragment paired end sequencing has yielded a wealth of single nucleotide variants and internal sequencing read insertions-deletions, at the cost of limited SV detection. Multi-kilobase DNA fragment mate pair sequencing has supplemented the void in SV detection, but introduced new analytic challenges requiring SV detection tools specifically designed for mate pair sequencing data. Here, we introduce SVachra – Structural Variation Assessment of CHRomosomal Aberrations, a breakpoint calling program that identifies large insertions-deletions, inversions, inter- and intra-chromosomal translocations utilizing both inward and outward facing read types generated by mate pair sequencing.ResultsWe demonstrate SVachra’s utility by executing the program on large-insert (Illumina Nextera) mate pair sequencing data from the personal genome of a single subject (HS1011). An additional data set of long-read (Pacific BioSciences RSII) was also generated to validate SV calls from SVachra and other comparison SV calling programs. SVachra exhibited the highest validation rate and reported the widest distribution of SV types and size ranges when compared to other SV callers.ConclusionsSVachra is a highly specific breakpoint calling program that exhibits a more unbiased SV detection methodology than other callers.
Highlights
Characterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing
The main restriction of such sequencing libraries is the contamination of inward facing reads, unbiotinylated fragments which map in an opposite orientation and smaller fragment size that confound the calling of chromosomal rearrangements by introducing contradictory discordant read information
SVachra was evaluated on a 6.2Kb average insert Nextera Tagmentation mate pair library of the HS1011 human genome that contains a causative single nucleotide variant for an autosomal recessive Charcot-Marie-Tooth neuropathy [2, 11, 12]
Summary
Characterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing. We introduce SVachra – Structural Variation Assessment of CHRomosomal Aberrations, a breakpoint calling program that identifies large insertions-deletions, inversions, inter- and intra-chromosomal translocations utilizing both inward and outward facing read types generated by mate pair sequencing. To alleviate the shortcomings of small fragment paired-end libraries, multi-kilobase mate pair and Nextera Tagmentation sequencing libraries have been introduced For these long-range mate pair libraries, fragments (usually on the order of 3-10Kb) are isolated, end labeled with biotin and circularized. The circularized molecules are sheared and the library is enriched for biotin labeled junction fragments Sequencing of these biotinylated fragments generates ‘outward-facing’ paired reads, meaning they align to the reference sequence in an outward facing direction from each other and at a distance in line with the selected long-range fragment size. The main restriction of such sequencing libraries is the contamination of inward facing reads, unbiotinylated fragments which map in an opposite orientation and smaller fragment size (usually between 200 and 300 bp) that confound the calling of chromosomal rearrangements by introducing contradictory discordant read information
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