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Mutational sequencing for accurate count and long-range assembly

Vijay Kumar, Julie Rosenbaum, Zihua Wang, Talitha Forcier, View ORCID ProfileMichael Ronemus, Michael Wigler, Dan Levy
doi: https://doi.org/10.1101/149740
Vijay Kumar
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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Julie Rosenbaum
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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Zihua Wang
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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Talitha Forcier
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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Michael Ronemus
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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  • ORCID record for Michael Ronemus
Michael Wigler
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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Dan Levy
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 USA
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ABSTRACT

We introduce a new protocol, mutational sequencing or muSeq, which randomly deaminates 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 a 135,000 fragment 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 13, 2017.
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Mutational sequencing for accurate count and long-range assembly
Vijay Kumar, Julie Rosenbaum, Zihua Wang, Talitha Forcier, Michael Ronemus, Michael Wigler, Dan Levy
bioRxiv 149740; doi: https://doi.org/10.1101/149740
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Mutational sequencing for accurate count and long-range assembly
Vijay Kumar, Julie Rosenbaum, Zihua Wang, Talitha Forcier, Michael Ronemus, Michael Wigler, Dan Levy
bioRxiv 149740; doi: https://doi.org/10.1101/149740

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