Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE75457 | GEO | 2015/12/04
SECONDARY ACCESSION(S): PRJNA304345
REPOSITORIES: GEO
Similar Datasets
Project description:We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells or mouse liver using different library prepartion methods. Libraries were preapred using MIN6 or Mouse Liver total RNA using (1) Illumina v1.5 small RNA library prep kit, (2) Illumina TruSeq kit, or (3) Bioo Scientific NEXTFlexV2
2015-12-04 | E-GEOD-75457 | biostudies-arrayexpress
Project description: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.
| S-EPMC4686641 | biostudies-literature
Project description:Comparison of library preparation methods using Cryptococcus neoformans var. grubii
| PRJEB7411 | ENA
Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.
2014-06-24 | E-MTAB-2566 | biostudies-arrayexpress
Project description:We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells cultured in 25mM glucose. We then developed and implemented an in-house short-read mapping strategy to analyze isomiR diversity. Profile of miRNA expression in MIN6 cells cultured in 25mM glucose.
2013-09-10 | E-GEOD-44262 | biostudies-arrayexpress
Project description:BackgroundNext-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.ResultsA pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias.ConclusionsThe single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.
| S-EPMC4117970 | biostudies-literature
Project description:We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells cultured in 25mM glucose. We then developed and implemented an in-house short-read mapping strategy to analyze isomiR diversity.
2013-09-10 | GSE44262 | GEO
Project description:The next-generation-sequencing (NGS) of ribosome footprintings and poly(A) enriched RNA-seq have been proformed to examine transcriptional and translational changes in an mouse insulinoma cell MIN6 under acute and chronic endoplasmic reticular stresses.
2021-05-20 | GSE174679 | GEO
Project description:We report performance of six different protocols for small RNAseq library preparation and of a method utilizing sequencing of probes targeting microRNAs (HTG EdgeSeq). Recently, small RNA sequencing (small RNA-seq) has been introduced as a method for quantifying circulating microRNAs (miRNAs) and enabling their global profiling without prior knowledge of target sequences. Despite its great promise, small RNA-seq has not delivered the expected outcomes, particularly due to ligation and PCR bias introduced within the workflow. In this study, we assessed the performance of all existing approaches to the small RNA-seq of miRNAs in plasma samples: original two adapter ligation approach; single adapter ligation with subsequent circularization; polyadenylation; use of randomized adapters; and use of unique molecular identifiers (UMI). Using comprehensive set of metrics, we evaluated each protocol in terms of yield, precision, accuracy, sensitivity, and ability to detect isomiRs. Moreover, we assessed performance of targeted RNA-seq method utilizing hybridization probes across relevant metrics and together with RT-qPCR we used it as a reference for accuracy evaluation. The best results were delivered by targeted RNA-seq outperforming other methods in all relevant parameters. The protocols using randomized adapters or UMIs showed consistent good performance across all of the assessed metrics. In contrast, the polyadenylation approach generated a high percentage of discarded reads and impeded the analysis of isomiRs. The single adapter ligation with subsequent circularization failed to prevent ligation bias and the traditional two adapter ligation approach achieved the worse scores in the majority of tested metrics. To sum, we provide a comprehensive comparison that can serve as a guide for new users interested in analysis of circulating miRNAs and as a reference for further comparative studies.
2020-12-31 | GSE149513 | GEO
Project description:Streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. Protocol is based on direct ligation of a fluorescently labeled adapter to the 3’end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation time from 4-5 to 2 days and increases sensitivity by 10-100-fold.
2020-12-29 | GSE163925 | GEO