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Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

Overview of attention for article published in Frontiers in Genetics, December 2015
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  • Good Attention Score compared to outputs of the same age (77th percentile)
  • High Attention Score compared to outputs of the same age and source (81st percentile)

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7 X users
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1 Facebook page
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1 Google+ user

Citations

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143 Mendeley
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Title
Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods
Published in
Frontiers in Genetics, December 2015
DOI 10.3389/fgene.2015.00352
Pubmed ID
Authors

Jeanette Baran-Gale, C. Lisa Kurtz, Michael R. Erdos, Christina Sison, Alice Young, Emily E. Fannin, Peter S. Chines, Praveen Sethupathy

Abstract

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5' and 3' ends of small RNAs. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study.

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X Demographics

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Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 143 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Spain 2 1%
Canada 1 <1%
Germany 1 <1%
Belgium 1 <1%
United States 1 <1%
Unknown 137 96%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 39 27%
Researcher 37 26%
Student > Master 13 9%
Student > Bachelor 11 8%
Student > Doctoral Student 7 5%
Other 15 10%
Unknown 21 15%
Readers by discipline Count As %
Agricultural and Biological Sciences 50 35%
Biochemistry, Genetics and Molecular Biology 44 31%
Medicine and Dentistry 8 6%
Engineering 5 3%
Computer Science 4 3%
Other 10 7%
Unknown 22 15%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 6. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 07 January 2016.
All research outputs
#6,101,028
of 24,520,935 outputs
Outputs from Frontiers in Genetics
#1,738
of 13,225 outputs
Outputs of similar age
#90,454
of 400,476 outputs
Outputs of similar age from Frontiers in Genetics
#9
of 43 outputs
Altmetric has tracked 24,520,935 research outputs across all sources so far. This one has received more attention than most of these and is in the 74th percentile.
So far Altmetric has tracked 13,225 research outputs from this source. They receive a mean Attention Score of 3.7. This one has done well, scoring higher than 86% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 400,476 tracked outputs that were published within six weeks on either side of this one in any source. This one has done well, scoring higher than 77% of its contemporaries.
We're also able to compare this research output to 43 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 81% of its contemporaries.