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Evaluating bias-reducing protocols for RNA sequencing library preparation.

ABSTRACT: BACKGROUND:Next-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. RESULTS: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 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. CONCLUSIONS:The 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.

SUBMITTER: Jackson TJ

PROVIDER: S-EPMC4117970 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Evaluating bias-reducing protocols for RNA sequencing library preparation.

Jackson Thomas J TJ Spriggs Ruth V RV Burgoyne Nicholas J NJ Jones Carolyn C Willis Anne E AE

BMC genomics 20140707

<h4>Background</h4>Next-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 sing ...[more]

PMID: 25001197

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Project description:BackgroundMicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored.ResultsWe assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar.ConclusionsThe sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.

Project description:Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27% (Fusobacterium nucleatum) to 73% (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n = 7) and a local outbreak of Klebsiella aerogenes (n = 5). The number of protocol steps and time required were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

Project description:Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here, we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a PCR step (Nextera), and two were PCR free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at -80°C) were assessed. All DNA isolations were performed in duplicate, totaling 80 samples, excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generating comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously. IMPORTANCE Previous research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.

Dataset Information

Evaluating bias-reducing protocols for RNA sequencing library preparation.

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Evaluating bias-reducing protocols for RNA sequencing library preparation.

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