Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing
Abstract Background Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-01-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-017-4428-5 |
| _version_ | 1811263188261404672 |
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| author | Laura E. MacConaill Robert T. Burns Anwesha Nag Haley A. Coleman Michael K. Slevin Kristina Giorda Madelyn Light Kevin Lai Mirna Jarosz Matthew S. McNeill Matthew D. Ducar Matthew Meyerson Aaron R. Thorner |
| author_facet | Laura E. MacConaill Robert T. Burns Anwesha Nag Haley A. Coleman Michael K. Slevin Kristina Giorda Madelyn Light Kevin Lai Mirna Jarosz Matthew S. McNeill Matthew D. Ducar Matthew Meyerson Aaron R. Thorner |
| author_sort | Laura E. MacConaill |
| collection | DOAJ |
| description | Abstract Background Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Results Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. Conclusions For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications. |
| first_indexed | 2024-04-12T19:40:09Z |
| format | Article |
| id | doaj.art-e5c8f64160a944ba9d2804d883e83001 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-12T19:40:09Z |
| publishDate | 2018-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-e5c8f64160a944ba9d2804d883e830012022-12-22T03:19:07ZengBMCBMC Genomics1471-21642018-01-0119111010.1186/s12864-017-4428-5Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencingLaura E. MacConaill0Robert T. Burns1Anwesha Nag2Haley A. Coleman3Michael K. Slevin4Kristina Giorda5Madelyn Light6Kevin Lai7Mirna Jarosz8Matthew S. McNeill9Matthew D. Ducar10Matthew Meyerson11Aaron R. Thorner12Center for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCIIntegrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Integrated DNA Technologies, Inc.Center for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCICenter for Cancer Genome Discovery, DFCIAbstract Background Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Results Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. Conclusions For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.http://link.springer.com/article/10.1186/s12864-017-4428-5Next generation sequencingMassively parallel sequencingAdapterIndexBarcode cross-talkIllumina |
| spellingShingle | Laura E. MacConaill Robert T. Burns Anwesha Nag Haley A. Coleman Michael K. Slevin Kristina Giorda Madelyn Light Kevin Lai Mirna Jarosz Matthew S. McNeill Matthew D. Ducar Matthew Meyerson Aaron R. Thorner Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing BMC Genomics Next generation sequencing Massively parallel sequencing Adapter Index Barcode cross-talk Illumina |
| title | Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing |
| title_full | Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing |
| title_fullStr | Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing |
| title_full_unstemmed | Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing |
| title_short | Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing |
| title_sort | unique dual indexed sequencing adapters with umis effectively eliminate index cross talk and significantly improve sensitivity of massively parallel sequencing |
| topic | Next generation sequencing Massively parallel sequencing Adapter Index Barcode cross-talk Illumina |
| url | http://link.springer.com/article/10.1186/s12864-017-4428-5 |
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