scholarly journals Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

2018 ◽  
Author(s):  
Carrie Wright ◽  
Anandita Rajpurohit ◽  
Emily E. Burke ◽  
Courtney Williams ◽  
Leonardo Collado-Torres ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Small Rna Sequencing ◽  
Library Preparation ◽  
Adapter Ligation ◽  
Improve Accuracy ◽  
Reliable Quantification

ABSTRACTHigh-throughput sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of the biases associated with small RNA sequencing have focused on adapter ligation bias with limited evaluation of reverse transcription or amplification biases. Furthermore, evaluations of the accuracy of quantifications of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited and no study has yet evaluated differences in the quantification of isomiRs of altered length. In addition, no studies have yet compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Kasey C Vickers ◽  
Michael G Levin ◽  
Michael P Anderson ◽  
Qing Xu ◽  
Joshua Anzinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Cellular Response ◽  
Culture Media ◽  
Recipient Cell ◽  
Inflammatory Cells ◽  
High Density Lipoproteins ◽  
Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

scholarly journals Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing

2013 ◽  
Author(s):  
Jeanette Baran-Gale ◽  
Michael R Erdos ◽  
Christina Sison ◽  
Alice Young ◽  
Emily E Fannin ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Library Preparation ◽  
Sequencing Technology ◽  
Preparation Methods ◽  
Sequencing Platforms ◽  
Small Rna Library ◽  
Illumina Library

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. Two widely used protocols for small RNA library preparation, Illumina v1.5 and Illumina TruSeq, use different pairs of adapter sequences. In this study, we compare the results of small RNA-sequencing between v1.5 and TruSeq and observe a striking differential bias. Nearly 100 highly expressed microRNAs (miRNAs) are >5-fold differentially detected and 48 miRNAs are >10-fold differentially detected between the two methods of library preparation. In fact, some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. The results are reproducible across different sequencing centers (NIH and UNC) and both major Illumina sequencing platforms, GAIIx and HiSeq. 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. As increasingly more laboratories transition to the newer TruSeq-based library preparation for small RNAs, researchers should be aware of the extent to which the results may differ from previously published results using v1.5.

scholarly journals Excess primer degradation by Exo I improves the preparation of 3′ cDNA ligation-based sequencing libraries

BioTechniques ◽  
2019 ◽  
Vol 67 (3) ◽  
pp. 110-116
Author(s):  
Christel Hougård Enroth ◽  
Annaleigh Ohrt Fehler ◽  
Line Dahl Poulsen ◽  
Jeppe Vinther
Keyword(s):  
Escherichia Coli ◽  
Rna Sequencing ◽  
Small Rna ◽  
Reverse Transcription ◽  
Library Construction ◽  
Reverse Transcription Primer ◽  
Adapter Ligation ◽  
Sequencing Library

RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3′ ends of cDNAs often produces primer–adapter byproducts, which compete with cDNA–adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer–adapter product contamination in 3′ cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer–adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3′ end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3′ cDNA ligation for sequencing-based applications.

scholarly journals A low-bias and sensitive small RNA library preparation method using randomized splint ligation

2020 ◽  
Vol 48 (14) ◽  
pp. e80-e80 ◽  
Author(s):  
Sean Maguire ◽  
Gregory J S Lohman ◽  
Shengxi Guan
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Preparation Process ◽  
Library Preparation ◽  
Normal Tissues ◽  
Levels Of Detail ◽  
Low Sensitivity ◽  
Next Generation Sequencing Ngs

Abstract Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.

Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories

2013 ◽  
Vol 31 (11) ◽  
pp. 1015-1022 ◽  
Author(s):  
Peter A C 't Hoen ◽  
◽  
Marc R Friedländer ◽  
Jonas Almlöf ◽  
Michael Sammeth ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Small Rna Sequencing

scholarly journals Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol C

2018 ◽  
Author(s):  
Roger P. Alexander ◽  
MD Giraldez ◽  
RM Spengler ◽  
A Etheridge ◽  
PM Godoy ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Library Preparation

scholarly journals Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol B

2018 ◽  
Author(s):  
Roger P. Alexander ◽  
MD Giraldez ◽  
RM Spengler ◽  
A Etheridge ◽  
PM Godoy ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Library Preparation

scholarly journals Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol A

2018 ◽  
Author(s):  
Roger P. Alexander ◽  
MD Giraldez ◽  
RM Spengler ◽  
A Etheridge ◽  
PM Godoy ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Library Preparation
Sign in / Sign up

Export Citation Format

Share Document