The impact of quality filter for RNA-Seq

Sá, Pablo Henrique Caracciolo Gomes de; Veras, Adonney Allan de Oliveira; Carneiro, Adriana Ribeiro; Pinheiro, Kenny C.; Pinto, Anne C.; Soares, Siomar C.; Schneider, Maria Paula Cruz; Azevedo, Vasco; Silva, Artur M. S.; Ramos, Rommel Thiago Jucá

doi:10.1016/j.gene.2015.03.033

Cited by 9 publications

(8 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The depth of coverage was adequate for the needs of the study according to Haas et al (2012) . Sequencing error rates should have occurred in the present study ( Wang et al, 2012 ); however, there was no attempt to eliminate those tRFs with few sequences ( de Sá et al, 2015 ). tRFs with low number of sequences could potentially be spurious results and should be taken into account when interpreting results from this study.…”

Section: Discussionmentioning

confidence: 85%

Characterization of circulating transfer RNA-derived RNA fragments in cattle

2015

View full text Add to dashboard Cite

The objective was to characterize naturally occurring circulating transfer RNA-derived RNA fragments (tRFs) in cattle1. Serum from eight clinically normal adult dairy cows was collected, and small non-coding RNAs were extracted immediately after collection and sequenced by Illumina MiSeq. Sequences aligned to transfer RNA (tRNA) genes or their flanking sequences were characterized. Sequences aligned to the beginning of 5′ end of the mature tRNA were classified as tRF5; those aligned to the 3′ end of mature tRNA were classified as tRF3; and those aligned to the beginning of the 3′ end flanking sequences were classified as tRF1. There were 3,190,962 sequences that mapped to transfer RNA and small non-coding RNAs in the bovine genome. Of these, 2,323,520 were identified as tRF5s, 562 were tRF3s, and 81 were tRF1s. There were 866,799 sequences identified as other small non-coding RNAs (microRNA, rRNA, snoRNA, etc.) and were excluded from the study. The tRF5s ranged from 28 to 40 nucleotides; and 98.7% ranged from 30 to 34 nucleotides in length. The tRFs with the greatest number of sequences were derived from tRNA of histidine, glutamic acid, lysine, glycine, and valine. There was no association between number of codons for each amino acid and number of tRFs in the samples. The reason for tRF5s being the most abundant can only be explained if these sequences are associated with function within the animal.

show abstract

Section: Discussionmentioning

confidence: 85%

Characterization of circulating transfer RNA-derived RNA fragments in cattle

2015

View full text Add to dashboard Cite

show abstract

“…With the rapid increase in the quality of RNASeq data over the past several years and the use of technical and biologic replicates, this next-generation sequencing approach will likely soon be thought of to have the same reliability as RT-PCR experiments, the current gold standard for gene expression evaluation (de Sa et al, 2015). While RT-PCR can only be utilized to assess gene expression one gene at a time, RNAseq allows for global transcriptome analysis.…”

Section: Discussionmentioning

confidence: 99%

RNAseq Reveals Complex Response of Campylobacter jejuni to Ovine Bile and In vivo Gallbladder Environment

et al. 2017

View full text Add to dashboard Cite

Colonization of the gallbladder by enteric pathogens such as Salmonella typhi, Listeria monocytogenes, and Campylobacter jejuni is thought to play a key role in transmission and persistence of these important zoonotic agents; however, little is known about the molecular mechanisms that allow for bacterial survival within this harsh environment. Recently, a highly virulent C. jejuni sheep abortion (SA) clone represented by the clinical isolate IA3902 has emerged as the dominant cause for sheep abortion in the United States. Previous studies have indicated that the C. jejuni clone SA can frequently be isolated from the gallbladders of otherwise healthy sheep, suggesting that the gallbladder may serve as an important reservoir for infection. To begin to understand the molecular mechanisms associated with survival in the host gallbladder, C. jejuni IA3902 was exposed for up to 24 h to both the natural ovine host in vivo gallbladder environment, as well as ovine bile in vitro. Following exposure, total RNA was isolated from the bile and high throughput deep sequencing of strand specific rRNA-depleted total RNA was used to characterize the transcriptome of IA3902 under these conditions. Our results demonstrated for the first time the complete transcriptome of C. jejuni IA3902 during exposure to an important host environment, the sheep gallbladder. Exposure to the host environment as compared to in vitro bile alone provided a more robust picture of the complexity of gene regulation required for survival in the host gallbladder. A subset of genes including a large number of protein coding genes as well as seven previously identified non-coding RNAs were confirmed to be differentially expressed within our data, suggesting that they may play a key role in adaptation upon exposure to these conditions. This research provides valuable insights into the molecular mechanisms that may be utilized by C. jejuni IA3902 to colonize and survive within the inhospitable gallbladder environment.

show abstract

“… Similarly, the choice of phred quality values is a trade-off between not excluding too many raw reads but retaining as many as possible good quality reads [ 77 ]. The phred-value impacts stronger on resulting quality of RNASeq data than on DNA based genome sequencing and is shown to possibly affect later gene expression results [ 82 ]. Illumina data should be filtered with a phred value of 30 or more, a phred value of 30 allows for an error rate of 99.9% (one erroneous base per 1000 bases can still be a lot depending on the sequence depth).…”

Section: Transcriptome Analysis and Its Complexitymentioning

confidence: 99%

Studying Smaller and Neglected Organisms in Modern Evolutionary Venomics Implementing RNASeq (Transcriptomics)—A Critical Guide

Reumont

2018

Toxins

View full text Add to dashboard Cite

Venoms are evolutionary key adaptations that species employ for defense, predation or competition. However, the processes and forces that drive the evolution of venoms and their toxin components remain in many aspects understudied. In particular, the venoms of many smaller, neglected (mostly invertebrate) organisms are not characterized in detail, especially with modern methods. For the majority of these taxa, even their biology is only vaguely known. Modern evolutionary venomics addresses the question of how venoms evolve by applying a plethora of -omics methods. These recently became so sensitive and enhanced that smaller, neglected organisms are now more easily accessible to comparatively study their venoms. More knowledge about these taxa is essential to better understand venom evolution in general. The methodological core pillars of integrative evolutionary venomics are genomics, transcriptomics and proteomics, which are complemented by functional morphology and the field of protein synthesis and activity tests. This manuscript focuses on transcriptomics (or RNASeq) as one toolbox to describe venom evolution in smaller, neglected taxa. It provides a hands-on guide that discusses a generalized RNASeq workflow, which can be adapted, accordingly, to respective projects. For neglected and small taxa, generalized recommendations are difficult to give and conclusions need to be made individually from case to case. In the context of evolutionary venomics, this overview highlights critical points, but also promises of RNASeq analyses. Methodologically, these concern the impact of read processing, possible improvements by perfoming multiple and merged assemblies, and adequate quantification of expressed transcripts. Readers are guided to reappraise their hypotheses on venom evolution in smaller organisms and how robustly these are testable with the current transcriptomics toolbox. The complementary approach that combines particular proteomics but also genomics with transcriptomics is discussed as well. As recently shown, comparative proteomics is, for example, most important in preventing false positive identifications of possible toxin transcripts. Finally, future directions in transcriptomics, such as applying 3rd generation sequencing strategies to overcome difficulties by short read assemblies, are briefly addressed.

show abstract

The impact of quality filter for RNA-Seq

Abstract: Even high-accuracy sequencing technologies are subject to the influence of quality filters when evaluating RNA-Seq data using the reference approach.

Cited by 9 publications

References 22 publications

Characterization of circulating transfer RNA-derived RNA fragments in cattle

Characterization of circulating transfer RNA-derived RNA fragments in cattle

RNAseq Reveals Complex Response of Campylobacter jejuni to Ovine Bile and In vivo Gallbladder Environment

Studying Smaller and Neglected Organisms in Modern Evolutionary Venomics Implementing RNASeq (Transcriptomics)—A Critical Guide

Contact Info

Product

Resources

About