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Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations.

Abstract：

:We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Fu GK,Xu W,Wilhelmy J,Mindrinos MN,Davis RW,Xiao W,Fodor SP

doi

10.1073/pnas.1323732111

subject

Has Abstract

pub_date

2014-02-04 00:00:00

pages

1891-6

issue

5

eissn

0027-8424

issn

1091-6490

pii

1323732111

journal_volume

111

pub_type

杂志文章

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