Bioo Scientific - Reduced Bias Small RNA Library Prep with Gel-Free...

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Reduced Bias Small RNA
Library Prep with Gel-Free
or Low-Input Options
HERD TOGETHER.
®

Small RNA sequencing is inherently
saddled with three distinct PROBLEMS!

Substantial bias introduced during ligation steps
Need to gel purify final library
Lack of low-input protocols
PROBLEM #
1...

PROBLEM #
1
Research shows extensive bias is typically
seen in small RNA sequencing
Baran-Gale, J., Kurtz, L. C., Erdos, S...

What causes this bias?

Research shows these inconsistencies are
primarily caused by bias introduced during
the ligation step of library prep
Jaya...

What is the SOLUTION?

Published research shows randomized
adapters reduce ligation bias
Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidan...

Bioo Scientific makes the only small RNA
sequencing kits for Illumina platforms that
use randomized adapters to reduce lig...

The NEXTflex Small RNA-Seq Kit v3
shows more equal coverage of an equimolar
pool of 24 miRNAs (miRNA calibrator),
demonstr...

Figure 1. Sequencing results from small RNA libraries created in triplicate from 1 ng of miRNA
Calibrator, an equimolar mi...

The NEXTflex Small RNA-Seq Kit v3
shows more even coverage with the Miltenyi
miRXplore Universal References, an
equimolar ...

Figure 2. Sequencing results from small RNA libraries created in triplicate from 1 ng of
Miltenyi miRXplore Universal Refe...

The use of randomized adapters in the
NEXTflex Small RNA-Seq Kit v3 greatly
improves accuracy of data by reducing
bias in ...

The NEXTflex Small RNA-Seq Kit v3
allows detection of more miRNAs in
total RNA samples

Figure 3. Small RNA libraries were created in duplicate from human brain total RNA and
sequenced on an Illumina MiSeq. The...

The NEXTflex Small RNA-Seq Kit v3
detects the same number of miRNAs
with fewer reads

Figure 4. 3 - 4.5x fewer reads are necessary to detect 100 miRNAs with
the NEXTflex Small RNA Seq-Kit v3.
Reads necessary ...

Reduced bias small RNA library prep
using the NEXTflex Small RNA-Seq Kit
v3 allows detection of more small RNAs
at lower s...

PROBLEM #
2
Small RNA library preparation has historically
required PAGE gel purification due to the
presence of adapter-d...

PAGE purification is tedious &
time consuming

PAGE purification limits throughput &
prevents start-to-finish automation

Elimination of adapter-dimer products allows
gel-free purification of small RNA libraries

The NEXTflex Small RNA-Seq Kit v3 uses
a dual approach to substantially reduce
adapter-dimer formation, allowing gel-free
...

Gel-free libraries prepared with the
NEXTflex Small RNA-Seq Kit v3 have
a higher proportion of reads mapping
to miRNAs

Figure 5. Percent of total reads aligned to miRBase in gel-free libraries
created from 100 ng human brain total RNA input....

Enhanced reduction of adapter-dimer
formation allows gel-free library preparation
CONCLUSION #
3

PROBLEM #
3
Lack of low-input protocols

Substantial reduction of
adapter-dimers also allows low
input library preparation

Libraries can be prepared from
as little as 1 ng total RNA

Low input libraries are achieved
by using up to 25 cycles of PCR

PCR bias adds negligible bias to
small RNA libraries

Correlation of miRNA expression
demonstrates negligible bias added by
additional PCR cycles
PCR Cycles 12 16 20 24 28
12 1...

Published data shows that additional
PCR cycles add negligible bias
to small RNA libraries
Jayaprakash, A.D., et al., Iden...

Expression values are reproducible across
different sample inputs
Figure 6. Correlation of miRNA expression between sample...

Gel purification of low-input small
RNA libraries is still necessary

Enhanced reduction of adapter-dimer
formation allows library preparation
from as little as 1 ng of total RNA
CONCLUSION #
4

The NEXTflex Small RNA-Seq Kit v3 is the
only commercially available kit that includes
randomized adapters to reduce bias ...

NEXTflex™ Small RNA-Seq Kit v3
(Illumina® Compatible)
• 48 unique barcodes for multiplexing included with 48 reaction kit
...

Learn more about the NEXTflex™ Small
RNA-Seq Kit v3 to obtain more accurate
results from small RNA sequencing
NEXTflex™
Sm...

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

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