Evaluating bias-reducing protocols for RNA sequencing library preparation
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REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR498210.fastq.gz | Fastqsanger.gz | |||
| ERR498211.fastq.gz | Fastqsanger.gz | |||
| ERR498212.fastq.gz | Fastqsanger.gz |
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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: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.
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Project description:Evaluating different library preparation methods for Pool-seq
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Project description:We performed a parallel analysis of commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se generates pronounced sequencing biases, and subsequent fragmentation and amplification steps lead to several-fold overrepresentation of these artefacts. Standard pre-bisulfite library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of methylation levels. We have integrated a bias diagnostic tool in the Bismark package and propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation.
2017-02-16 | GSE77961 | GEO
Project description:gnp07_regeneome_microdissectionbias - microdissection vs no microdissection - Transcriptome bias induce by protocols used in microdissection - To compare RNA from flowers and RNA from microdissected flowers to know bias induce by microdissection (dissection from all tissues were collected to have a representation of entire flower)
2012-06-01 | GSE30265 | GEO
Project description:gnp07_regeneome_microdissectionbias - microdissection vs no microdissection - Transcriptome bias induce by protocols used in microdissection - To compare RNA from flowers and RNA from microdissected flowers to know bias induce by microdissection (dissection from all tissues were collected to have a representation of entire flower) 2 dye-swap - treated vs untreated comparison
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Project description:RNA-Seq technique was applied to investigate the effects of four cDNA amplification kits and two RNA-Seq library preparation kits to the deep sequencing results at different perspectives.
2015-05-09 | GSE57503 | GEO
Project description:RNA-Seq technique was applied to investigate the effects of four cDNA amplification kits and two RNA-Seq library preparation kits to the deep sequencing results at different perspectives. The same set of semen samples were applied to investigate the qualitative and quantitative effect of four cDNA amplification methods and two RNA-Seq library preparation methods on sperm transcript profiling.
2015-05-09 | E-GEOD-57503 | biostudies-arrayexpress
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:The continuing ‘desire’ in obtaining more quantitative and detailed information from cellular proteomics experiments in regards to proteome coverage and protein modifications asks for a systematic investigation of the protein extraction and digestion protocols, in particular when working with unicellular organisms with a strong cell wall such as the model yeast S. cerevisiae. The selection of a suitable sample preparation workflow is crucial to obtain in-depth proteome coverage, as the use of certain sample preparation protocols may bias the protein identification or induce (unexpected) peptide modifications. Here, we made an extensive comparison of preparation workflows commonly applied to S. cerevisiae. Workflows were compared on the basis of identified MS/MS spectra, peptide sequences and number and type of modifications using both restricted (Peaks) and unrestricted (TagGraph) database search approaches. The proteome coverage was mainly affected by the sample collection approach, while it was maximized using a FASP. Extensive reagent specific peptide modifications were detected when using formic acid, but also when using acetone. Such artefacts split the analyte mass signals and generate additional chemical noise that may also elute differently compared to the native peptide. The use of both an unrestricted and restricted database search increased identification rates significantly and resulted in the identification of approximately 70% of the MS2 spectra for the best protocol. The unidentified spectra were assessed by their de novo sequencing score. This confirmed that those spectra consisted by a majority of very low quality spectra, insufficient to match to database sequences. However, a small fraction of the unidentified spectra showed high quality, which presumably derive from unknown sequence variants not present in the database. This study demonstrates the high importance of the sample preparation workflow and the obtained results will guide researchers in the field to optimize sample preparation procedures.
2022-03-31 | PXD026806 | Pride