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 #2

PROBLEM #3

PROBLEM #1Research shows extensive bias is typically

seen in small RNA sequencingBaran-Gale, J., Kurtz, L. C., Erdos, Sison, M. C., Young, A., Fannin, E. E., Chines, P. S. and Sethupathy, P. (2015) Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade

capture by current methods. Frontiers in Genetics. doi: 10.3389/fgene.2015.00352.

What causes this bias?

Research shows these inconsistencies are primarily caused by bias introduced during

the ligation step of library prep

Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidanandam, R. (Sept 2, 2011), Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing Nuc Acid Res, 1–12. doi:10.1093/nar/gkr693

Zhang, et al. (2013) High-efficiency cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biology 14 R109.

Sun, G. (2011) A bias-reducing strategy in profiling small RNAs using Solexa. RNA. 17: 2256-2262

Sorefan, K. et al. (2012) Reducing sequencing bias of small RNAs. Silence. doi:10.1186/1758-907X-3-4

What is the SOLUTION?

Published research shows randomized adapters reduce ligation bias

Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidanandam, R. (Sept 2, 2011), Identification and remediation of biases in the activity of RNA ligases in small-RNA

deep sequencing Nuc Acid Res, 1–12. doi:10.1093/nar/gkr693.

Bioo Scientific makes the only small RNA sequencing kits for Illumina platforms that

use randomized adapters to reduce ligase bias

The NEXTflex Small RNA-Seq Kit v3 shows more equal coverage of an equimolar

pool of 24 miRNAs (miRNA calibrator), demonstrating more accurate representation

of the original sample

Figure 1. Sequencing results from small RNA libraries created in triplicate from 1 ng of miRNA Calibrator, an equimolar mixture of 24 miRNAs. Values farther from 1 indicate more bias.

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CVNEXTflex = 1.09Illumina = 2.35

NEB = 2.31

NEXTflex Illumina NEB

The NEXTflex Small RNA-Seq Kit v3 shows more even coverage with the Miltenyi

miRXplore Universal References, an equimolar mixture of 963 miRNAs

Figure 2. Sequencing results from small RNA libraries created in triplicate from 1 ng of Miltenyi miRXplore Universal Reference, an equimolar mixture of 963 miRNAs.

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CVNEXTflex = 1.14Illumina = 3.78

NEB = 1.67

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

bias in small RNA library prep

CONCLUSION #1

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 indicated number of reads was sampled from each library

and the average number of miRNA groups with ≥20 reads determined. The inset shows the number of reads required to detect 100 miRNA groups at a threshold of ≥20 reads.

Sequencing depth versus miRNAs detected

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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 withthe NEXTflex Small RNA Seq-Kit v3.

Reads necessary todetect 100 miRNAs

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

at lower sequencing depth

CONCLUSION #2

PROBLEM #2Small RNA library preparation has historically

required PAGE gel purification due to the presence of adapter-dimer products

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 purification of final libraries

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.

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

CONCLUSION #3

PROBLEM #3Lack 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 cyclesPCR Cycles 12 16 20 24 28

12 1 0.9999 0.9994 0.9966 0.980716 1 0.9996 0.9970 0.981320 1 0.9975 0.982424 1 0.981628 1

Table 1. Correlation of miRNA abundance in libraries created using serial 10x dilutions of cDNA and the indicated number of PCR cycles. The Pearson correlation coefficients

calculated from the Log10(reads) values of miRNAs with ≥ 10 reads in all samples are shown

Published data shows that additional PCR cycles add negligible bias

to small RNA librariesJayaprakash, A.D., et al., Identification and remediation of biases in the activity of

RNA ligases in small-RNA deep sequencing. Nucleic Acids Res, 2011. 39(21): p. e141.

Hafner, M., et al., RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA, 2011. 17(9): p. 1697-712.

Expression values are reproducible across different sample inputs

Figure 6. Correlation of miRNA expression between samples created with 100 ng and 10 ng of human brain total RNA with the NEXTflex Small RNA-Seq kit v3. The Pearson correlation

coefficient is shown.

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R = 0.964

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 and allows gel-free or low-input library prep

NEXTflex™ Small RNA-Seq Kit v3(Illumina® Compatible)

• 48 unique barcodes for multiplexing included with 48 reaction kit

• 8 reaction kit includes barcodes that allow low-level multiplexing

Learn more about the NEXTflex™ Small RNA-Seq Kit v3 to obtain more accurate

results from small RNA sequencing

NEXTflex™ Small RNA-Seq Kit v3(Illumina Compatible)

Catalog #: 5132-05 (8 reactions)

GEL-FREE & LOW INPUT OPTIONS

©Bioo Scienti�c Corporation · 2015 V15.11

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nextgen@biooscientific.com THE NGS EXPERTS™

Reduced-Bias Small RNA Library Preparation with Gel-Free or Low-Input Options

INTRODUCTION

Changes in microRNA (miRNA) expression have been shown to be associated with a variety of normal physiological processes, as well as diseases including cancer. Studies have already shown that miRNAs may provide useful markers for the development of disease

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Bias is introduced into small RNA libraries during the ligation steps. Small RNA library preparation involves ligating adapters directly onto the 3’ and 5’ ends of the RNA molecule in two separate steps, and each of these ligation steps has been shown to introduce severe bias into library preparation. Recent studies have shown that inserting random bases at the ends of the adapters greatly reduces bias in comparison to using non-randomized adapters [1-4].

-ration. In typical DNA or RNA library prep, insert-containing molecules are at least 100 bp larger than adapter-dimer molecules, and thus can be removed using SPRI magnetic beads. However, since insert-containing molecules are only ~20 bp larger than adapter-dimer

size selection greatly limits both throughput and automation potential of small RNA library preparation, as only a limited number of libraries can be run on a single gel and it is a labor-intensive process that is not amenable to automation.

unique in that additional PCR cycles result in negligible bias; thus it should theoretically be possible to create low-input small RNA

products and leading to sequencing data where very few of the reads are useful. A number of methods have been developed to reduce

such an extent that gel-free or low-input small RNA library preparation is possible.

method that utilizes randomized adapters for greatly reduced bias and features gel-free or low-input protocols, which are possible due .

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