Partial bisulfite conversion for unique template sequencing

Kumar, V., Rosenbaum, J., Wang, Z., Forcier, T., Ronemus, M., Wigler, M., Levy, D. (January 2018) Partial bisulfite conversion for unique template sequencing. Nucleic Acids Res, 46 (2). e10. ISSN 0305-1048

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URL: https://www.ncbi.nlm.nih.gov/pubmed/29161423
DOI: 10.1093/nar/gkx1054

Abstract

We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone.

Item Type: Paper
Subjects: Investigative techniques and equipment > assays > next generation sequencing
CSHL Authors:
Communities: CSHL labs > Kinney lab
CSHL labs > Levy lab
CSHL labs > Wigler lab
CSHL Cancer Center Program > Cancer Genetics
Depositing User: Matt Covey
Date: 25 January 2018
Date Deposited: 01 Dec 2017 21:11
Last Modified: 12 Jun 2018 16:14
PMCID: PMC5778454
Related URLs:
URI: http://repository.cshl.edu/id/eprint/35679

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