Handling multi-mapped reads in RNA-seq

Deschamps-Francoeur, Gabrielle; Simoneau, Joël; Scott, Michelle S.

doi:10.1016/j.csbj.2020.06.014

Cited by 65 publications

(52 citation statements)

References 56 publications

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“…This feature allows better distinction of paralogous genes (Deschamps-Francoeur et al, 2020), which, for example, are present at high degrees in all teleost species, due to their shared whole genome duplication event (Glasauer and Neuhauss, 2014). Handling multi-mapped reads has been shown to increase alignment accuracy, resulting in better transcript detection and quantification (Deschamps-Francoeur et al, 2020). One of the most popular approaches to correctly count multi-mapped reads is expectation maximization, which has been included already in several tools like RSEM (Li and Dewey, 2011), Salmon (Patro et al, 2017) as well as kallisto (Bray et al, 2016;Melsted et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets

Shainer

Stemmer

2021

Preprint

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Background: Single-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard downstream processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics Cell Ranger pipeline. Results: In most of the tested samples, kallisto outperformed Cell Ranger in sequencing read alignment rates and total gene detection rates. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and processing pipeline can affect the discovery of biologically-relevant cell types. Conclusion: While Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. It is thus beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.

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Section: Discussionmentioning

confidence: 99%

Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets

Shainer

Stemmer

2021

Preprint

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“…Bowtie2 version 2.3.4.3 ( 83 ) was used to align the subsequent sets of reads to all the rRNA and tRNA loci from the T. vaginalis genome, retaining only unaligned reads and read pairs. Finally, the sRNA-Seq data sets were collapsed to unique sequences as per best practices for genomes that contain multiple repeats ( 84 ) using the clumpify tool from the BBMap package version 37.48 ( 85 ) and used in all subsequent analyses. The number of reads remaining after each filtering step is shown in Table 3 .…”

Section: Methodsmentioning

confidence: 99%

Small RNAs Are Implicated in Regulation of Gene and Transposable Element Expression in the Protist Trichomonas vaginalis

Warring

Blow

Avecilla

et al. 2021

mSphere

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Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent nonviral sexually transmitted infection worldwide. Repetitive elements, including transposable elements (TEs) and virally derived repeats, comprise more than half of the ∼160-Mb T. vaginalis genome. An intriguing question is how the parasite controls its potentially lethal complement of mobile elements, which can disrupt transcription of protein-coding genes and genome functions. In this study, we generated high-throughput RNA sequencing (RNA-Seq) and small RNA-Seq data sets in triplicate for the T. vaginalis G3 reference strain and characterized the mRNA and small RNA populations and their mapping patterns along all six chromosomes. Mapping the RNA-Seq transcripts to the genome revealed that the majority of genes predicted within repetitive elements are not expressed. Interestingly, we identified a novel species of small RNA that maps bidirectionally along the chromosomes and is correlated with reduced protein-coding gene expression and reduced RNA-Seq coverage in repetitive elements. This novel small RNA family may play a regulatory role in gene and repetitive element expression. Our results identify a possible small RNA pathway mechanism by which the parasite regulates expression of genes and TEs and raise intriguing questions as to the role repeats may play in shaping T. vaginalis genome evolution and the diversity of small RNA pathways in general. IMPORTANCE Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most common nonviral sexually transmitted infection in humans. The millions of cases each year have sequelae that may include complications during pregnancy and increased risk of HIV infection. Given its evident success in this niche, it is paradoxical that T. vaginalis harbors in its genome thousands of transposable elements that have the potential to be extremely detrimental to normal genomic function. In many organisms, transposon expression is regulated by the activity of endogenously expressed short (∼21 to 35 nucleotides [nt]) small RNA molecules that effect gene silencing by targeting mRNAs for degradation or by recruiting epigenetic silencing machinery to locations in the genome. Our research has identified small RNA molecules correlated with reduced expression of T. vaginalis genes and transposons. This suggests that a small RNA pathway is a major contributor to gene expression patterns in the parasite and opens up new avenues for investigation into small RNA biogenesis, function, and diversity.

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“…A total of 371 patients were included for the study including 121 female and 250 male patients. RNA-Seq by Expectation-Maximization (RSEM) ( 13 ) was used for accurate transcript abundance quantification, and the resulting values were used for subsequent statistical analysis. The age cut-off was set as 55: Young (aged <55 years) and old patients (aged ≥55 years).…”

Section: Methodsmentioning

confidence: 99%

Long non‑coding RNA BACE1‑AS is an independent unfavorable prognostic factor in liver cancer

Nie

et al. 2020

Oncol Lett

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Liver cancer is one of the leading causes of cancer-associated deaths with incidence rates continuously on the rise. Biomarkers are urgently required for early diagnosis and better prognostic classification, which is essential for risk stratification and optimizing treatment strategies in clinical settings. By analyzing the data extracted from The Cancer Genome Atlas database using R, the long noncoding RNA (lncRNA) β-site APP-cleaving enzyme 1 antisense (BACE1-AS) was discovered to have both high diagnostic and prognostic values in liver cancer, which could serve as a promising biomarker in clinical settings. Precisely, lncRNA BACE1-AS is significantly overexpressed in liver cancer and its levels vary within different subgroups, suggesting its tumorigenic role. Furthermore, higher BACE1-AS predicts poorer overall survival and relapse-free survival outcomes. Overall, the present study demonstrated that BACE1-AS may be involved in liver cancer progression and could serve as a promising biomarker for diagnosis and prognostic evaluation.

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Handling multi-mapped reads in RNA-seq

Cited by 65 publications

References 56 publications

Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets

Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets

Small RNAs Are Implicated in Regulation of Gene and Transposable Element Expression in the Protist Trichomonas vaginalis

Long non‑coding RNA BACE1‑AS is an independent unfavorable prognostic factor in liver cancer

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