Structural Variant genotyping by using short-read massive parallel sequencing of restriction DNA circles

Abstract

Background: Structural variants (SVs) are large DNA rearrangements, typically >50 bp, that may be directly involved in genome evolution and human diseases. SVs can be classified as copy number variants (CNVs), characterized by gain (tandem duplications and insertions) or loss of genetic material (deletions); non-CNVs, large rearrangements non involving gain or loss of DNA (perfect inversions, reciprocal translocations), and complex, combining characteristics. The Human Genome (HG) presents a vast complexity characterized by a high number of long and particularly short interspersed repeated elements (LINEs and SINEs, respectively) as well as low copy number repeats (LCRs) (typically 2-4 copies per haploid genome). Homologous pairing and recombination between non-allelic copies of these repeats may fuel the occurrence of all type of SVs. These SVs mediated by unequal homologous recombination are characterized by being delimited by these repeats and, consequently, the specific breakpoint cannot be determined and will remain undefined and confined to the range of the repeated tract involved in the reciprocal. Massive parallel sequencing (MPS) has greatly improved modern human genetics, so called genomic medicine, by mean of performing a comprehensive and accurate genotyping of small variants including single nucleotide substitutions and indels (SNV). However, SV genotyping by using 2nd generation MPS (e.g., Illumina platform of short read sequencing) and the associated bioinformatics algorithms are still far to reach its potential. 3rd generation MPS has come to address the challenges posed by SV genotyping by producing much longer reads. It is expected that long-read MPS will alleviate numerous computational challenges surrounding genome assembly, transcript reconstruction, and metagenomics among other important areas of modern biology and medicine. Among 2nd generation MPS, pair-end read technologies, is highly accurate for SNV genotyping, but no so efficient for SV calling in the complex HG, in part due to its abundance in repeated sequences such as SINE, LINE and LCR. Certainty, long read sequencing technologies or third generation MPS, have clear advantages for SV genotyping, but its insertion in genomic medicine worldwide is still. Consequently, some recurrent SVs, mediated by large LCRs (e.g., >1 kb), still need characterization by massive sequencing. Aim: The objective of this work is to present the proof-of-concept of a novel approach for SV genotyping characterized by applying second generation Whole Genome Sequencing (WGS) from circularized restriction-fragment DNA and the development of the specifically designed bioinformatic protocol.