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

Wang M, Beck CR, English AC, Meng Q, Buhay C, Han Y, Doddapaneni HV, Yu F, Boerwinkle E, Lupski JR, Muzny DM, Gibbs RA - BMC Genomics (2015)

Bottom Line: PacBio-LITS provides deep sequence coverage at the specified sites at substantially reduced cost compared with PacBio WGS.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.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, 77030, USA. mwang@bcm.edu.

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.

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Workflow of the PacBio-LITS method.A. sample intake QC; B. DNA fragmentation; C. target size selection using Pippin/BluePippin; D. construction of pre-capture library using size-selected DNA fragments; E. Roche/NimbleGen SeqCap EZ solution-based target enrichment using custom probes; F. construction of PacBio library using post-capture product and G. PacBio long read-length sequencing.
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Fig1: Workflow of the PacBio-LITS method.A. sample intake QC; B. DNA fragmentation; C. target size selection using Pippin/BluePippin; D. construction of pre-capture library using size-selected DNA fragments; E. Roche/NimbleGen SeqCap EZ solution-based target enrichment using custom probes; F. construction of PacBio library using post-capture product and G. PacBio long read-length sequencing.

Mentions: The new method consists of two major steps: 1) large-insert capture library preparation, and 2) PacBio library preparation using the captured product as template (Figure 1). The sample intake QC step involves analysis by agarose gel electrophoresis to determine DNA integrity (e.g. intact or degraded) and measuring the sample concentration by PicoGreen dsDNA assay or Qubit fluorometric quantitation. DNA fragmentation is achieved by using Covaris Focused-ultrasonicator or a g-TUBE apparatus. Selection for the targeted insert size is performed using Sage Science’s Pippin (for 1 Kb insert) or BluePippin (for >1 Kb insert) platform under specific running parameters. The size-selected DNA fragments then undergo a pre-capture library preparation process that is similar to the standard Illumina paired-end library construction involving end repair, 3′-adenylation, adaptor ligation and ligation-mediated PCR (LM-PCR). Target enrichment of the pre-capture library DNA follows the Roche/NimbleGen liquid hybridization protocol using specific solution probes (SeqCap probes). The post-capture product serves as the input DNA for PacBio large-insert library preparation. The final product, a large insert capture library with PacBio SMRT bell adaptors ligated to both ends of the inserts, is loaded onto the PacBio platform for long read-length sequencing. A complete protocol for 6 Kb insert PacBio capture library constructions is appended (see Additional file 1: BCM-HGSC PacBio-LITS Protocol).Figure 1


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

Wang M, Beck CR, English AC, Meng Q, Buhay C, Han Y, Doddapaneni HV, Yu F, Boerwinkle E, Lupski JR, Muzny DM, Gibbs RA - BMC Genomics (2015)

Workflow of the PacBio-LITS method.A. sample intake QC; B. DNA fragmentation; C. target size selection using Pippin/BluePippin; D. construction of pre-capture library using size-selected DNA fragments; E. Roche/NimbleGen SeqCap EZ solution-based target enrichment using custom probes; F. construction of PacBio library using post-capture product and G. PacBio long read-length sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4376517&req=5

Fig1: Workflow of the PacBio-LITS method.A. sample intake QC; B. DNA fragmentation; C. target size selection using Pippin/BluePippin; D. construction of pre-capture library using size-selected DNA fragments; E. Roche/NimbleGen SeqCap EZ solution-based target enrichment using custom probes; F. construction of PacBio library using post-capture product and G. PacBio long read-length sequencing.
Mentions: The new method consists of two major steps: 1) large-insert capture library preparation, and 2) PacBio library preparation using the captured product as template (Figure 1). The sample intake QC step involves analysis by agarose gel electrophoresis to determine DNA integrity (e.g. intact or degraded) and measuring the sample concentration by PicoGreen dsDNA assay or Qubit fluorometric quantitation. DNA fragmentation is achieved by using Covaris Focused-ultrasonicator or a g-TUBE apparatus. Selection for the targeted insert size is performed using Sage Science’s Pippin (for 1 Kb insert) or BluePippin (for >1 Kb insert) platform under specific running parameters. The size-selected DNA fragments then undergo a pre-capture library preparation process that is similar to the standard Illumina paired-end library construction involving end repair, 3′-adenylation, adaptor ligation and ligation-mediated PCR (LM-PCR). Target enrichment of the pre-capture library DNA follows the Roche/NimbleGen liquid hybridization protocol using specific solution probes (SeqCap probes). The post-capture product serves as the input DNA for PacBio large-insert library preparation. The final product, a large insert capture library with PacBio SMRT bell adaptors ligated to both ends of the inserts, is loaded onto the PacBio platform for long read-length sequencing. A complete protocol for 6 Kb insert PacBio capture library constructions is appended (see Additional file 1: BCM-HGSC PacBio-LITS Protocol).Figure 1

Bottom Line: PacBio-LITS provides deep sequence coverage at the specified sites at substantially reduced cost compared with PacBio WGS.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.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, 77030, USA. mwang@bcm.edu.

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.

Show MeSH
Related in: MedlinePlus