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Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.

Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C - Nat. Biotechnol. (2009)

Bottom Line: We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out.About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper.One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

View Article: PubMed Central - PubMed

Affiliation: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. gnirke@broad.mit.edu

ABSTRACT
Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

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Related in: MedlinePlus

Overview of hybrid selection method. Illustrated are steps involved in the preparation of a complex pool of biotinylated RNA capture probes (“bait”; top left), whole-genome fragment input library (“pond”; top right) and hybrid-selected enriched output library (“catch”; bottom). Two sequencing targets and their respective baits are shown in red and blue. Thin and thick lines represent single and double strands, respectively. Universal adapter sequences are grey. The excess of single-stranded non-self-complementary RNA (wavy lines) drives the hybridization. See main text and Methods for details.
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Figure 1: Overview of hybrid selection method. Illustrated are steps involved in the preparation of a complex pool of biotinylated RNA capture probes (“bait”; top left), whole-genome fragment input library (“pond”; top right) and hybrid-selected enriched output library (“catch”; bottom). Two sequencing targets and their respective baits are shown in red and blue. Thin and thick lines represent single and double strands, respectively. Universal adapter sequences are grey. The excess of single-stranded non-self-complementary RNA (wavy lines) drives the hybridization. See main text and Methods for details.

Mentions: We developed a method for capturing sequencing targets that combines the flexibility and economy of oligonucleotide synthesis on a microarray with the favorable kinetics of hybridization in solution (Fig. 1). A complex pool of ultra-long 200-mer oligonucleotides is synthesized in parallel on an Agilent microarray and then cleaved from the array. Each oligonucleotide consists of a target-specific 170-mer sequence flanked by 15 bases of a universal primer sequence on each side to allow PCR amplification. After the initial PCR, a T7 promoter is added in a second round of PCR. We then use in vitro transcription in the presence of biotin-UTP to generate a single-stranded RNA hybridization “bait” for “fishing” targets of interest out of a “pond” of randomly sheared, adapter-ligated and PCR-amplified total human DNA. The hybridization is driven by the vast excess of RNA baits that cannot self-anneal. The “catch” is pulled-down with streptavidin-coated magnetic beads, PCR-amplified with universal primers, and analyzed on a “next-generation” sequencing instrument. The method allows preparation of large amounts of bait from a single oligonucleotide array synthesis that can be quality control tested, stored in aliquots and used repeatedly over the course of a large-scale targeted sequencing project.


Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.

Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C - Nat. Biotechnol. (2009)

Overview of hybrid selection method. Illustrated are steps involved in the preparation of a complex pool of biotinylated RNA capture probes (“bait”; top left), whole-genome fragment input library (“pond”; top right) and hybrid-selected enriched output library (“catch”; bottom). Two sequencing targets and their respective baits are shown in red and blue. Thin and thick lines represent single and double strands, respectively. Universal adapter sequences are grey. The excess of single-stranded non-self-complementary RNA (wavy lines) drives the hybridization. See main text and Methods for details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663421&req=5

Figure 1: Overview of hybrid selection method. Illustrated are steps involved in the preparation of a complex pool of biotinylated RNA capture probes (“bait”; top left), whole-genome fragment input library (“pond”; top right) and hybrid-selected enriched output library (“catch”; bottom). Two sequencing targets and their respective baits are shown in red and blue. Thin and thick lines represent single and double strands, respectively. Universal adapter sequences are grey. The excess of single-stranded non-self-complementary RNA (wavy lines) drives the hybridization. See main text and Methods for details.
Mentions: We developed a method for capturing sequencing targets that combines the flexibility and economy of oligonucleotide synthesis on a microarray with the favorable kinetics of hybridization in solution (Fig. 1). A complex pool of ultra-long 200-mer oligonucleotides is synthesized in parallel on an Agilent microarray and then cleaved from the array. Each oligonucleotide consists of a target-specific 170-mer sequence flanked by 15 bases of a universal primer sequence on each side to allow PCR amplification. After the initial PCR, a T7 promoter is added in a second round of PCR. We then use in vitro transcription in the presence of biotin-UTP to generate a single-stranded RNA hybridization “bait” for “fishing” targets of interest out of a “pond” of randomly sheared, adapter-ligated and PCR-amplified total human DNA. The hybridization is driven by the vast excess of RNA baits that cannot self-anneal. The “catch” is pulled-down with streptavidin-coated magnetic beads, PCR-amplified with universal primers, and analyzed on a “next-generation” sequencing instrument. The method allows preparation of large amounts of bait from a single oligonucleotide array synthesis that can be quality control tested, stored in aliquots and used repeatedly over the course of a large-scale targeted sequencing project.

Bottom Line: We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out.About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper.One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

View Article: PubMed Central - PubMed

Affiliation: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. gnirke@broad.mit.edu

ABSTRACT
Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Show MeSH
Related in: MedlinePlus