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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

Coverage profiles of exon targets by end sequencing and shotgun sequencing. Shown are cumulative coverage profiles that sum the per-base sequencing coverage along 7,052 single-bait target exons. Only free-standing baits that were not within 500 bases of another one were included in this analysis. End sequencing of exon capture 1 with 36-base reads (a) produced a bimodal profile with high sequence coverage near and slightly beyond the ends of the 170-base baits (indicated by the horizontal bar). Shotgun sequencing of capture 2 from a different pond library (containing fragments with generic rather than Illumina-specific adapters) with 36-base reads after concatenating and re-shearing (b) gave more coverage on bait (shaded area) than near bait. Re-sequencing of capture 1 with 76-base end reads (c) had a similar effect, although the peak was slightly wider and the on-bait fraction of the peak area slightly less. Note that the scale on the Y-axis and hence the absolute peak height is different in each case. The different scales reflect the different numbers of sequenced bases which is much lower for GA-I lanes (a, b) than for a GA-II lane (c).
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Figure 2: Coverage profiles of exon targets by end sequencing and shotgun sequencing. Shown are cumulative coverage profiles that sum the per-base sequencing coverage along 7,052 single-bait target exons. Only free-standing baits that were not within 500 bases of another one were included in this analysis. End sequencing of exon capture 1 with 36-base reads (a) produced a bimodal profile with high sequence coverage near and slightly beyond the ends of the 170-base baits (indicated by the horizontal bar). Shotgun sequencing of capture 2 from a different pond library (containing fragments with generic rather than Illumina-specific adapters) with 36-base reads after concatenating and re-shearing (b) gave more coverage on bait (shaded area) than near bait. Re-sequencing of capture 1 with 76-base end reads (c) had a similar effect, although the peak was slightly wider and the on-bait fraction of the peak area slightly less. Note that the scale on the Y-axis and hence the absolute peak height is different in each case. The different scales reflect the different numbers of sequenced bases which is much lower for GA-I lanes (a, b) than for a GA-II lane (c).

Mentions: The high stringency of hybridization selects for fragments that contain a substantial portion of the bait sequence. As a result, fragments with both ends near or outside the ends of the bait sequence are overrepresented relative to fragments that have less overlap and thus end near the middle. Merely end-sequencing the fragments with short 36-base reads therefore leads to elevated coverage near the end of the baits, with many reads falling outside the target, and a pronounced dip in coverage in the center. This effect is evident in the cumulative coverage profile representing 7,052 free-standing single-bait targets (Fig. 2a).


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)

Coverage profiles of exon targets by end sequencing and shotgun sequencing. Shown are cumulative coverage profiles that sum the per-base sequencing coverage along 7,052 single-bait target exons. Only free-standing baits that were not within 500 bases of another one were included in this analysis. End sequencing of exon capture 1 with 36-base reads (a) produced a bimodal profile with high sequence coverage near and slightly beyond the ends of the 170-base baits (indicated by the horizontal bar). Shotgun sequencing of capture 2 from a different pond library (containing fragments with generic rather than Illumina-specific adapters) with 36-base reads after concatenating and re-shearing (b) gave more coverage on bait (shaded area) than near bait. Re-sequencing of capture 1 with 76-base end reads (c) had a similar effect, although the peak was slightly wider and the on-bait fraction of the peak area slightly less. Note that the scale on the Y-axis and hence the absolute peak height is different in each case. The different scales reflect the different numbers of sequenced bases which is much lower for GA-I lanes (a, b) than for a GA-II lane (c).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Coverage profiles of exon targets by end sequencing and shotgun sequencing. Shown are cumulative coverage profiles that sum the per-base sequencing coverage along 7,052 single-bait target exons. Only free-standing baits that were not within 500 bases of another one were included in this analysis. End sequencing of exon capture 1 with 36-base reads (a) produced a bimodal profile with high sequence coverage near and slightly beyond the ends of the 170-base baits (indicated by the horizontal bar). Shotgun sequencing of capture 2 from a different pond library (containing fragments with generic rather than Illumina-specific adapters) with 36-base reads after concatenating and re-shearing (b) gave more coverage on bait (shaded area) than near bait. Re-sequencing of capture 1 with 76-base end reads (c) had a similar effect, although the peak was slightly wider and the on-bait fraction of the peak area slightly less. Note that the scale on the Y-axis and hence the absolute peak height is different in each case. The different scales reflect the different numbers of sequenced bases which is much lower for GA-I lanes (a, b) than for a GA-II lane (c).
Mentions: The high stringency of hybridization selects for fragments that contain a substantial portion of the bait sequence. As a result, fragments with both ends near or outside the ends of the bait sequence are overrepresented relative to fragments that have less overlap and thus end near the middle. Merely end-sequencing the fragments with short 36-base reads therefore leads to elevated coverage near the end of the baits, with many reads falling outside the target, and a pronounced dip in coverage in the center. This effect is evident in the cumulative coverage profile representing 7,052 free-standing single-bait targets (Fig. 2a).

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