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Orthogonal NGS for High Throughput Clinical Diagnostics.

Chennagiri N, White EJ, Frieden A, Lopez E, Lieber DS, Nikiforov A, Ross T, Batorsky R, Hansen S, Lip V, Luquette LJ, Mauceli E, Margulies D, Milos PM, Napolitano N, Nizzari MM, Yu T, Thompson JF - Sci Rep (2016)

Bottom Line: This approach yields genomic scale orthogonal confirmation of ~95% of exome variants.Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other.We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

View Article: PubMed Central - PubMed

Affiliation: Claritas Genomics, Cambridge MA, USA.

ABSTRACT
Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

No MeSH data available.


Related in: MedlinePlus

Comparison of per-exon coverage achieved on NextSeq and Proton platforms.Mean coverage for each exome was normalized to 100×. Coverage for each exon was plotted on a log scale with exons having no reads changed to 1× for plotting. Dashed lines show 20× coverage for each platform.
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f1: Comparison of per-exon coverage achieved on NextSeq and Proton platforms.Mean coverage for each exome was normalized to 100×. Coverage for each exon was plotted on a log scale with exons having no reads changed to 1× for plotting. Dashed lines show 20× coverage for each platform.

Mentions: First, we analyzed how well each sequencing approach covered our analytic target. For comparison purposes, NextSeq and Proton data was numerically normalized to a mean depth of 100×. In Fig. 1, mean coverage on both platforms for each exon is plotted for all 187,475 exons in our analytic region as a function of sequencing method. Exons with no coverage were adjusted up to 1× in order to allow log-log plotting. The graph was split into four quadrants based on mean 20× coverage. The great majority of exons (>90%) were covered by at least 20 reads by both platforms. 4327 (2.3%) exons failed to achieve at least 20× coverage on both platforms with 2253 (1.2%) of these having less than 10× coverage on both platforms. More than 8% of exons were well covered (>20× mean coverage) by one platform but not the other (4.7% or 8892 with >20× coverage on NextSeq only and 3.7% or 6973 with >20× coverage on Proton only). Many of the exons found on only the NextSeq or only the Proton are difficult to sequence and thus NIST has not included them in their reference. Because they are not in NIST, they do not impact the apparent sensitivity listed in Table 2. Thus, use of two orthogonal platforms improves the orthogonal sensitivity ~3–4% relative to the use of one platform alone. This estimate is based on the number of exons where variants can be detected on only one platform.


Orthogonal NGS for High Throughput Clinical Diagnostics.

Chennagiri N, White EJ, Frieden A, Lopez E, Lieber DS, Nikiforov A, Ross T, Batorsky R, Hansen S, Lip V, Luquette LJ, Mauceli E, Margulies D, Milos PM, Napolitano N, Nizzari MM, Yu T, Thompson JF - Sci Rep (2016)

Comparison of per-exon coverage achieved on NextSeq and Proton platforms.Mean coverage for each exome was normalized to 100×. Coverage for each exon was plotted on a log scale with exons having no reads changed to 1× for plotting. Dashed lines show 20× coverage for each platform.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836299&req=5

f1: Comparison of per-exon coverage achieved on NextSeq and Proton platforms.Mean coverage for each exome was normalized to 100×. Coverage for each exon was plotted on a log scale with exons having no reads changed to 1× for plotting. Dashed lines show 20× coverage for each platform.
Mentions: First, we analyzed how well each sequencing approach covered our analytic target. For comparison purposes, NextSeq and Proton data was numerically normalized to a mean depth of 100×. In Fig. 1, mean coverage on both platforms for each exon is plotted for all 187,475 exons in our analytic region as a function of sequencing method. Exons with no coverage were adjusted up to 1× in order to allow log-log plotting. The graph was split into four quadrants based on mean 20× coverage. The great majority of exons (>90%) were covered by at least 20 reads by both platforms. 4327 (2.3%) exons failed to achieve at least 20× coverage on both platforms with 2253 (1.2%) of these having less than 10× coverage on both platforms. More than 8% of exons were well covered (>20× mean coverage) by one platform but not the other (4.7% or 8892 with >20× coverage on NextSeq only and 3.7% or 6973 with >20× coverage on Proton only). Many of the exons found on only the NextSeq or only the Proton are difficult to sequence and thus NIST has not included them in their reference. Because they are not in NIST, they do not impact the apparent sensitivity listed in Table 2. Thus, use of two orthogonal platforms improves the orthogonal sensitivity ~3–4% relative to the use of one platform alone. This estimate is based on the number of exons where variants can be detected on only one platform.

Bottom Line: This approach yields genomic scale orthogonal confirmation of ~95% of exome variants.Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other.We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

View Article: PubMed Central - PubMed

Affiliation: Claritas Genomics, Cambridge MA, USA.

ABSTRACT
Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

No MeSH data available.


Related in: MedlinePlus