Investigation of chimeric reads using the MinION [version 2; peer review: 2 approved]
Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR...
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Published in | F1000 research Vol. 6; p. 631 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
London, UK
F1000Research
2017
F1000 Research Ltd |
Subjects | |
Online Access | Get full text |
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Summary: | Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading. Despite the separation of ligation products, chimeric reads formed from different amplicons were still observed in the base-called sequence. The long-read nature of nanopore sequencing presents an effective tool for the discovery and filtering of chimeric reads. We have found that at least 1.7% of reads prepared using the Nanopore LSK002 2D Ligation Kit include post-amplification chimeric elements. This finding has potential implications for other amplicon sequencing technologies, as the process is unlikely to be specific to the sample preparation used for nanopore sequencing. |
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Bibliography: | new_version ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing interests: The R9.4 flow cell and sequencing kit (SQK-LSK208) used for this experiment were provided free of charge by ONT as replacements for a purchased kit and flow cell where the phenomena of chimeric reads was initially discovered. ONT provided advice regarding the sample preparation protocols, including the suggestion of a slow overnight ligation step. RW: Sample preparation and QC; CP: Mouse injections, RNA extraction; FR: Project oversight; OL: Sample preparation, project design and oversight; DE: DNA sequencing and bioinformatics analysis. All authors contributed towards the preparation of the manuscript. |
ISSN: | 2046-1402 2046-1402 |
DOI: | 10.12688/f1000research.11547.2 |