PCR bias during RNA-seq library preparation can incorrectly amplify some transcripts over others, making accurate quantification of transcript numbers difficult. Molecular indexing uses unique molecular identifier adapters to label each transcript, correcting for PCR bias and improving RNA quantification. Published research shows molecular indexing returns over 15% of reads to libraries and improves accuracy, especially for highly expressed genes, by accounting for distinct fragments with identical start/stop sites eliminated by typical unique molecular identifier methods.

1. Absolute Quantitation
for RNA-Seq

2. RNA-Seq requires accurate
quantitation of transcript numbers,
which can be difficult to define

3. It’s difficult to define because of
PCR BIAS

4. The efficiency of PCR amplification
is sequence-dependent, with some
transcripts being preferentially
amplified over others

5. Conventional RNA-Seq data will
tell you how many reads were
obtained for any transcript

6. But not how many were in your
initial sample
mRNA
3 Read Pairs
Unknown Number
of Fragments
3’5’

7. How can PCR bias be corrected?

8. By randomly tagging each RNA
transcript with a unique Molecular
Index Adapter™ during the ligation
step of library prep

9. RNA
Fragmented RNA
1st
Strand Synthesis
2nd
Strand Synthesis
Adenylation
Ligation of Y Adapters
with Molecular Labels
Degradation of 2nd
Strand
PCR Amplification
Sequencing
3’
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3’
5’
5’ 3’
3’
5’
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3’
3’
3’ 5’
5’
5’
5’
3’ UA
A
U U U U
3’5’
5’
5’
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FORW
ARD
PRIM
ER
BARCODED
PRIM
ER
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Sequencing Primer
Sequencing Primer
Read 1
Read 2

10. Which produces a labeled cDNA
library prior to PCR amplification

11. After PCR amplification of the library
tagged with Molecular Index Adapters,
all RNA transcripts can be detected
mRNA
3 Read Pairs
1 Fragment
3 Read Pairs
2 Fragments
3 Read Pairs
3 Fragments
3’5’

12. Improving the accuracy and reliability
of RNA transcript quantitation

13. Typically, USS (unique start/stop)
is used to eliminate PCR duplicates,
eliminating all fragments with identical
start and stop sites

14. 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

15. 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.

16. 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.

17. Therefore the traditional USS method
can incorrectly eliminate unique
fragments from NGS data

18. 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.

19. The NEXTflex™ Rapid Directional
qRNA-Seq™ Kit contains Molecular
Index Adapters, enabling high precision
measurement of unique RNA-Seq reads

20. 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.

21. Bioo Scientific also offers a
complementary script to simplify
quantitative RNA-seq data analysis

22. This kit has now been automated on the
Sciclone NGS and NGSx Workstations
®
DOWNLOAD MANUAL

23. For more information about how direcitonal
RNA libraries prepared using Molecular
Index Adapters can improve your RNA
quantitation, download this whitepaper.
DOWNLOAD WHITEPAPER

24. If you have any questions email us at
nextgen@biooscientific.com
or visit our website at
BiooScientific.com/DirectionalqRNA