Dataset Information

Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level.

ABSTRACT: RNA sequencing (RNA-seq) is currently the standard method for genome-wide expression profiling. RNA-seq reads often need to be mapped to a reference genome before read counts can be produced for genes. Read trimming methods have been developed to assist read mapping by removing adapter sequences and low-sequencing-quality bases. It is however unclear what is the impact of read trimming on the quantification of RNA-seq data, an important task in RNA-seq data analysis. In this study, we used a benchmark RNA-seq dataset and simulation data to assess the impact of read trimming on mapping and quantification of RNA-seq reads. We found that adapter sequences can be effectively removed by read aligner via 'soft-clipping' and that many low-sequencing-quality bases, which would be removed by read trimming tools, were rescued by the aligner. Accuracy of gene expression quantification from using untrimmed reads was found to be comparable to or slightly better than that from using trimmed reads, based on Pearson correlation with reverse transcriptase-polymerase chain reaction data and simulation truth. Total data analysis time was reduced by up to an order of magnitude when read trimming was not performed. Our study suggests that read trimming is a redundant process in the quantification of RNA-seq expression data.

SUBMITTER: Liao Y

PROVIDER: S-EPMC7671312 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level.

Liao Yang Y Shi Wei W

NAR genomics and bioinformatics 20200903 3

RNA sequencing (RNA-seq) is currently the standard method for genome-wide expression profiling. RNA-seq reads often need to be mapped to a reference genome before read counts can be produced for genes. Read trimming methods have been developed to assist read mapping by removing adapter sequences and low-sequencing-quality bases. It is however unclear what is the impact of read trimming on the quantification of RNA-seq data, an important task in RNA-seq data analysis. In this study, we used a ben ...[more]

PMID: 33575617

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Project description:High-throughput RNA-Sequencing (RNA-Seq) has become the preferred technique for studying gene expression differences between biological samples and for discovering novel isoforms, though the techniques to analyze the resulting data are still immature. One pre-processing step that is widely but heterogeneously applied is trimming, in which low quality bases, identified by the probability that they are called incorrectly, are removed. However, the impact of trimming on subsequent alignment to a genome could influence downstream analyses including gene expression estimation; we hypothesized that this might occur in an inconsistent manner across different genes, resulting in differential bias.To assess the effects of trimming on gene expression, we generated RNA-Seq data sets from four samples of larval Drosophila melanogaster sensory neurons, and used three trimming algorithms--SolexaQA, Trimmomatic, and ConDeTri-to perform quality-based trimming across a wide range of stringencies. After aligning the reads to the D. melanogaster genome with TopHat2, we used Cuffdiff2 to compare the original, untrimmed gene expression estimates to those following trimming. With the most aggressive trimming parameters, over ten percent of genes had significant changes in their estimated expression levels. This trend was seen with two additional RNA-Seq data sets and with alternative differential expression analysis pipelines. We found that the majority of the expression changes could be mitigated by imposing a minimum length filter following trimming, suggesting that the differential gene expression was primarily being driven by spurious mapping of short reads. Slight differences with the untrimmed data set remained after length filtering, which were associated with genes with low exon numbers and high GC content. Finally, an analysis of paired RNA-seq/microarray data sets suggests that no or modest trimming results in the most biologically accurate gene expression estimates.We find that aggressive quality-based trimming has a large impact on the apparent makeup of RNA-Seq-based gene expression estimates, and that short reads can have a particularly strong impact. We conclude that implementation of trimming in RNA-Seq analysis workflows warrants caution, and if used, should be used in conjunction with a minimum read length filter to minimize the introduction of unpredictable changes in expression estimates.

Project description:BackgroundRNA-Seq has become increasingly popular in transcriptome profiling. One aspect of transcriptome research is to quantify the expression levels of genomic elements, such as genes, their transcripts and exons. Acquiring a transcriptome expression profile requires genomic elements to be defined in the context of the genome. Multiple human genome annotation databases exist, including RefGene (RefSeq Gene), Ensembl, and the UCSC annotation database. The impact of the choice of an annotation on estimating gene expression remains insufficiently investigated.ResultsIn this paper, we systematically characterized the impact of genome annotation choice on read mapping and transcriptome quantification by analyzing a RNA-Seq dataset generated by the Human Body Map 2.0 Project. The impact of a gene model on mapping of non-junction reads is different from junction reads. For the RNA-Seq dataset with a read length of 75 bp, on average, 95% of non-junction reads were mapped to exactly the same genomic location regardless of which gene models was used. By contrast, this percentage dropped to 53% for junction reads. In addition, about 30% of junction reads failed to align without the assistance of a gene model, while 10-15% mapped alternatively. There are 21,958 common genes among RefGene, Ensembl, and UCSC annotations. When we compared the gene quantification results in RefGene and Ensembl annotations, 20% of genes are not expressed, and thus have a zero count in both annotations. Surprisingly, identical gene quantification results were obtained for only 16.3% (about one sixth) of genes. Approximately 28.1% of genes' expression levels differed by 5% or higher, and of those, the relative expression levels for 9.3% of genes (equivalent to 2038) differed by 50% or greater. The case studies revealed that the gene definition differences in gene models frequently result in inconsistency in gene quantification.ConclusionsWe demonstrated that the choice of a gene model has a dramatic effect on both gene quantification and differential analysis. Our research will help RNA-Seq data analysts to make an informed choice of gene model in practical RNA-Seq data analysis.

Project description:Background High-throughput RNA-Sequencing (RNA-Seq) has become the preferred technique for studying gene expression differences between biological samples and for discovering novel isoforms, though the techniques to analyze the resulting data are still immature. One pre-processing step that is widely but heterogeneously applied is trimming, in which low quality bases, identified by the probability that they are called incorrectly, are removed. However, the impact of trimming on subsequent alignment to a genome could influence downstream analyses including gene expression estimation; we hypothesized that this might occur in an inconsistent manner across different genes, resulting in differential bias. Results To assess the effects of trimming on gene expression, we generated RNA-Seq data sets from four samples of larval Drosophila melanogaster sensory neurons, and used three trimming algorithmsâSolexaQA, Trimmomatic, and ConDeTriâto perform quality-based trimming across a wide range of stringencies. After aligning the reads to the D. melanogaster genome with TopHat2, we used Cuffdiff2 to compare the original, untrimmed gene expression estimates to those following trimming. With the most aggressive trimming parameters, over ten percent of genes had significant changes in their estimated expression levels. This trend was seen with two additional RNA-Seq data sets and with alternative differential expression analysis pipelines. We found that the majority of the expression changes could be mitigated by imposing a minimum length filter following trimming, suggesting that the differential gene expression was primarily being driven by spurious mapping of short reads. Slight differences with the untrimmed data set remained after length filtering, which were associated with genes with low exon numbers and high GC content. Finally, an analysis of paired RNA-seq/microarray data sets suggests that no or modest trimming results in the most biologically accurate gene expression estimates. Conclusions We find that aggressive quality-based trimming has a large impact on the apparent makeup of RNA-Seq-based gene expression estimates, and that short reads can have a particularly strong impact. We conclude that implementation of trimming in RNA-Seq analysis workflows warrants caution, and if used, should be used in conjunction with a minimum read length filter to minimize the introduction of unpredictable changes in expression estimates. Four biological replicates of 100 Drosophila melanogaster larval multi-dendritic sensory neurons were profiled by mRNA-Seq

Dataset Information

Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level.

Publications

Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level.

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