PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations.

 
TitlePacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations.
Publication TypeJournal Article
Year of Publication2015
AuthorsWang, M, Beck, CR, English, AC, Meng, Q, Buhay, C, Han, Y, Doddapaneni, H, Yu, F, Boerwinkle, E, Lupski, JR, Muzny, DM, Gibbs, RA
JournalBMC Genomics
Volume16
Pagination214
Date Published2015 Mar 19
ISSN1471-2164
KeywordsChromosome Aberrations, Gene Library, Gene Rearrangement, Genetic Association Studies, Genomics, High-Throughput Nucleotide Sequencing, Humans, Workflow
Abstract

BACKGROUND: Generation of long (>5 Kb) DNA sequencing reads provides an approach for interrogation of complex regions in the human genome. Currently, large-insert whole genome sequencing (WGS) technologies from Pacific Biosciences (PacBio) enable analysis of chromosomal structural variations (SVs), but the cost to achieve the required sequence coverage across the entire human genome is high.

RESULTS: We developed a method (termed PacBio-LITS) that combines oligonucleotide-based DNA target-capture enrichment technologies with PacBio large-insert library preparation to facilitate SV studies at specific chromosomal regions. PacBio-LITS provides deep sequence coverage at the specified sites at substantially reduced cost compared with PacBio WGS. The efficacy of PacBio-LITS is illustrated by delineating the breakpoint junctions of low copy repeat (LCR)-associated complex structural rearrangements on chr17p11.2 in patients diagnosed with Potocki-Lupski syndrome (PTLS; MIM#610883). We successfully identified previously determined breakpoint junctions in three PTLS cases, and also were able to discover novel junctions in repetitive sequences, including LCR-mediated breakpoints. The new information has enabled us to propose mechanisms for formation of these structural variants.

CONCLUSIONS: The new method leverages the cost efficiency of targeted capture-sequencing as well as the mappability and scaffolding capabilities of long sequencing reads generated by the PacBio platform. It is therefore suitable for studying complex SVs, especially those involving LCRs, inversions, and the generation of chimeric Alu elements at the breakpoints. Other genomic research applications, such as haplotype phasing and small insertion and deletion validation could also benefit from this technology.

DOI10.1186/s12864-015-1370-2
Alternate JournalBMC Genomics
PubMed ID25887218
PubMed Central IDPMC4376517
Grant ListU54HG003273 / HG / NHGRI NIH HHS / United States
U54 HG006542 / HG / NHGRI NIH HHS / United States
R01NS058529 / NS / NINDS NIH HHS / United States
U54 HD083092 / HD / NICHD NIH HHS / United States
U54 HG003273 / HG / NHGRI NIH HHS / United States
R01 NS058529 / NS / NINDS NIH HHS / United States
U54HD006542 / HD / NICHD NIH HHS / United States