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Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

Damiati E, Borsani G, Giacopuzzi E - Hum. Genet. (2016)

Bottom Line: Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively.The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively.False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

View Article: PubMed Central - PubMed

Affiliation: Unit of Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy.

ABSTRACT
The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

No MeSH data available.


Related in: MedlinePlus

AmpliSeq exome properties and target region analysis. a Length density distribution calculated from the dimension of amplicons generated with the AmpliSeq Exome kit as determined from the provided BED. In the kit design, most of the CDS exons are covered by a single amplicon (b). The comparison of target regions across 6 different enrichment kits (c) revealed that no one fully address all the human CDS nor relevant clinical genes. The bar graph represents the number of genes with at least one exon partially addressed (green) or completely missed (red). Compared exome capture kits include: AmpliSeq Exome and TargetSeq Exome (Life Technologies), SureSelect Human All Exon v5/v6 (Agilent Technologies) and SeqCap EZ v2/v3 (Roche)
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Fig1: AmpliSeq exome properties and target region analysis. a Length density distribution calculated from the dimension of amplicons generated with the AmpliSeq Exome kit as determined from the provided BED. In the kit design, most of the CDS exons are covered by a single amplicon (b). The comparison of target regions across 6 different enrichment kits (c) revealed that no one fully address all the human CDS nor relevant clinical genes. The bar graph represents the number of genes with at least one exon partially addressed (green) or completely missed (red). Compared exome capture kits include: AmpliSeq Exome and TargetSeq Exome (Life Technologies), SureSelect Human All Exon v5/v6 (Agilent Technologies) and SeqCap EZ v2/v3 (Roche)

Mentions: The AmpliSeq Exome kit (Life Technologies) uses a multiplex-PCR approach to simultaneously generate about 294,000 amplicons designed on CDS sequences of the human genome (Table 1). Size of amplicons ranges between 156 and 240 bp (Fig. 1a) and the kit design addresses 300,887 CDS exons, 61.5 % of whom are covered by a single amplicon (Fig. 1b). To better determine the actual content of target regions and the extent of non-addressed clinically relevant regions, we compared the target intervals of AmpliSeq Exome kit with those of other popular exome capture kits, namely Life Technologies TargetSeq, Agilent SureSelect and Roche SeqCap EZ Exome. As described in methods, we determined for each kit the fraction of CDS bases comprised in the design and extrapolated the number of genes not fully addressed, with particular interest on ClinVar pathological genes and the 56 genes included in the incidental findings recommended report by ACMG. AmpliSeq Exome target regions covered a high proportion of bases in human CDS (97.5 %), ClinVar genes (94.1 %) and ACMG genes (98.9 %). However, even this small fraction of missed bases resulted in several genes with at least one exon completely missed: 2041 (7 %), 449 (13 %) and 11 (19 %) genes show this issue in human CDS, ClinVar and ACMG gene list, respectively (Fig. 1c). An example of a gene not completely addressed is reported in Supplementary figure 1a. Detailed statistics calculated for each exome enrichment kit are reported in supplementary file 1 and the complete list of the exons not fully addressed is provided in supplementary tables 1–6.Table 1


Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

Damiati E, Borsani G, Giacopuzzi E - Hum. Genet. (2016)

AmpliSeq exome properties and target region analysis. a Length density distribution calculated from the dimension of amplicons generated with the AmpliSeq Exome kit as determined from the provided BED. In the kit design, most of the CDS exons are covered by a single amplicon (b). The comparison of target regions across 6 different enrichment kits (c) revealed that no one fully address all the human CDS nor relevant clinical genes. The bar graph represents the number of genes with at least one exon partially addressed (green) or completely missed (red). Compared exome capture kits include: AmpliSeq Exome and TargetSeq Exome (Life Technologies), SureSelect Human All Exon v5/v6 (Agilent Technologies) and SeqCap EZ v2/v3 (Roche)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835520&req=5

Fig1: AmpliSeq exome properties and target region analysis. a Length density distribution calculated from the dimension of amplicons generated with the AmpliSeq Exome kit as determined from the provided BED. In the kit design, most of the CDS exons are covered by a single amplicon (b). The comparison of target regions across 6 different enrichment kits (c) revealed that no one fully address all the human CDS nor relevant clinical genes. The bar graph represents the number of genes with at least one exon partially addressed (green) or completely missed (red). Compared exome capture kits include: AmpliSeq Exome and TargetSeq Exome (Life Technologies), SureSelect Human All Exon v5/v6 (Agilent Technologies) and SeqCap EZ v2/v3 (Roche)
Mentions: The AmpliSeq Exome kit (Life Technologies) uses a multiplex-PCR approach to simultaneously generate about 294,000 amplicons designed on CDS sequences of the human genome (Table 1). Size of amplicons ranges between 156 and 240 bp (Fig. 1a) and the kit design addresses 300,887 CDS exons, 61.5 % of whom are covered by a single amplicon (Fig. 1b). To better determine the actual content of target regions and the extent of non-addressed clinically relevant regions, we compared the target intervals of AmpliSeq Exome kit with those of other popular exome capture kits, namely Life Technologies TargetSeq, Agilent SureSelect and Roche SeqCap EZ Exome. As described in methods, we determined for each kit the fraction of CDS bases comprised in the design and extrapolated the number of genes not fully addressed, with particular interest on ClinVar pathological genes and the 56 genes included in the incidental findings recommended report by ACMG. AmpliSeq Exome target regions covered a high proportion of bases in human CDS (97.5 %), ClinVar genes (94.1 %) and ACMG genes (98.9 %). However, even this small fraction of missed bases resulted in several genes with at least one exon completely missed: 2041 (7 %), 449 (13 %) and 11 (19 %) genes show this issue in human CDS, ClinVar and ACMG gene list, respectively (Fig. 1c). An example of a gene not completely addressed is reported in Supplementary figure 1a. Detailed statistics calculated for each exome enrichment kit are reported in supplementary file 1 and the complete list of the exons not fully addressed is provided in supplementary tables 1–6.Table 1

Bottom Line: Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively.The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively.False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

View Article: PubMed Central - PubMed

Affiliation: Unit of Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy.

ABSTRACT
The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

No MeSH data available.


Related in: MedlinePlus