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. 2014 Feb 4;111(5):1891-6.
doi: 10.1073/pnas.1323732111. Epub 2014 Jan 21.

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

Glenn K Fu  1 , Weihong Xu, Julie Wilhelmy, Michael N Mindrinos, Ronald W Davis, Wenzhong Xiao, Stephen P A Fodor
Affiliations

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

Glenn K Fu et al. Proc Natl Acad Sci U S A. .

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.

Keywords: RNA-seq; cDNA library; molecular barcoding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Illustration of the addition of molecular indices to cDNA molecules. Each end of the 3′ adenylated cDNA fragment (light blue bars) ligates randomly to one of 96 possible 3′ T overhang A or B adaptors (purple or dark blue bars). On each adaptor, the molecular index is represented as an 8-nucleotide sequence barcode (yellow). cDNAs with ligated adaptors can be amplified by using PCR primers A and B. If desired, a sample index barcode (brown bar) can be added to primer A. (B) PCR-amplified clones can be sequenced by using primers corresponding to the adaptor sequences. (C) An outline of the experiment workflow is shown.
Fig. 2.
Fig. 2.
Library efficiency determination by direct spike-in of synthetic, barcoded, poly-adenylated RNA transcripts. Molecules of RNA are identical in sequence except for a 21-nt-long barcode sequence (molecular index) embedded within each transcript. A total of 960 barcodes were created, and each can be identified by RT-PCR amplification with dye-labeled PCR primers, followed by hybridization detection on an array of oligonucleotide probes. Arrows show the location of the PCR primers used.
See this image and copyright information in PMC

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