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Absolute Quantitationfor RNA-Seq
RNA-Seq requires accurate quantitation of transcript numbers,
which can be difficult to define
It’s difficult to define because of
PCR BIAS
The efficiency of PCR amplification is sequence-dependent, with some
transcripts being preferentially amplified over others
Conventional RNA-Seq data will tell you how many reads were
obtained for any transcript
But not how many were in your initial sample
mRNA
3 Read PairsUnknown Numberof Fragments
3’5’
How can PCR bias be corrected?
By randomly tagging each RNA transcript with a unique Molecular Index Adapter™ during the ligation
step of library prep
RNAFragmented RNA
1st Strand Synthesis2nd Strand Synthesis
Adenylation
Ligation of Y Adapterswith Molecular Labels
Degradation of 2nd Strand
PCR Amplification
Sequencing
3’
3’ U U U U U
U U U U U
3’
3’
5’
5’ 3’
3’
5’
5’
3’
3’3’ 5’
5’
5’5’
3’ UAA
U U U U3’5’5’
5’
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FORWARD PRIMERBARCODED PRIM
ER
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Sequencing Primer
Sequencing Primer
Read 1
Read 2
Which produces a labeled cDNA library prior to PCR amplification
After PCR amplification of the library tagged with Molecular Index Adapters,
all RNA transcripts can be detected
mRNA
3 Read Pairs1 Fragment
3 Read Pairs2 Fragments
3 Read Pairs3 Fragments
3’5’
Improving the accuracy and reliability of RNA transcript quantitation
Typically, USS (unique start/stop) is used to eliminate PCR duplicates,
eliminating all fragments with identical start and stop sites
However, far more often than expected, distinct fragments share
the same start/stop sites
Fu, G.K., Xu, W., Wilhelmy, J., Mindrinos, M.N., Davis, R.W., Xiao, W., and Fodor, S.P.A. 2013. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. PNAS.
111(5):1891-6. doi: 10.1073/pnas.1323732111
Molecular Labels combined with USS correction returned more than 15% of
reads to the libraries
0%
20%
40%
60%
80%
100%
qy2qy1dqy2dqy1
Unique Reads(all transcripts)
USS + STL USS
The number of unique fragments as determined by unique start and stop correction (USS) and a USS correction combined with molecular labels (USS + STL).
For more information read the whitepaper.
The percentage of reads corrected is higher in genes with high expression
0%
20%
40%
60%
80%
100%
qy2qy1dqy2dqy1
Unique Reads(transcripts with ≥ 1000 read pairs)
USS + STL USS
The number of unique fragments as determined by unique start and stop correction (USS) and a USS correction combined with molecular labels (USS + STL).
For more information read the whitepaper.
Therefore the traditional USS method can incorrectly eliminate unique
fragments from NGS data
Published research shows Molecular Index Adapters improve the accuracy and reliability
of RNA transcript quantitation
Fu, G.K., Xu, W., Wilhelmy, J., Mindrinos, M.N., Davis, R.W., Xiao, W., and Fodor, S.P.A. 2013. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. PNAS.
111(5):1891-6. doi: 10.1073/pnas.1323732111
Fu, G.K., Hu, J., Wang, P.H., and Fodor, S.P. 2011. Counting individual DNA molecules by the stochastic attachment of diverse labels. PNAS 108:9026-9031. doi: 10.1073/pnas.1017621108.
Shiroguchi, K., Jia, P.A. Sims, and Xie, X.S. 2012. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. PNAS 109:1347-1352. doi: 10.1073/pnas.1118018109.
Head, S.R., Komori, H.K., LaMere, S.A. et. al. 2014. Library construction for next-generation sequencing: Overviews and challenges. BioTechniques 56(2):61–77. doi: 10.2144/000114133.
The NEXTflex™ Rapid Directional qRNA-Seq™ Kit contains Molecular
Index Adapters, enabling high precision measurement of unique RNA-Seq reads
The NEXTflex Rapid Directional qRNA-Seq Kit is ideal for constructing stranded
Illumina RNA-Seq libraries from 10 - 100 ng of mRNA or rRNA-depleted RNA with >99%
strand specificity.
Bioo Scientific also offers a complementary script to simplify
quantitative RNA-seq data analysis
This kit has now been automated on the Sciclone NGS and NGSx Workstations
®
DOWNLOAD MANUAL
For more information about how direcitonal RNA libraries prepared using Molecular Index Adapters can improve your RNA quantitation, download this whitepaper.
DOWNLOAD WHITEPAPER
If you have any questions email us [email protected]
or visit our website atBiooScientific.com/DirectionalqRNA
