RNA-seq: technical variability and sampling

McIntyre, Lauren M.; Lopiano, Kenneth K.; Morse, Alison M.; Amin, V.; Oberg, Ann L.; Young, Linda J.; Nuzhdin, Sergey V.

doi:10.1186/1471-2164-12-293

Cited by 269 publications

(229 citation statements)

References 38 publications

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“…In similar studies of pigs (Chen et al, 2011;Esteve-Codina et al, 2011) and cattle (Huang et al, 2012), in which 61.6-77.8% of reads were matched to the reference genome, our results were comparable for sheep. The percentage of reads mapped to the reference genes were lower than that mapped to the O. aries genome, a result potentially due to the GC content, type of cells, and other reasons (McIntyre et al, 2011;Sendler et al, 2011). The percentage of reads mapped to the reference genes in the LCL libraries (59.91, 56.89, and 56.09%) were slightly higher than those for the MCL libraries (55.03, 56.44, and 54.87%), but the percentage of reads mapped to the O. aries genome were not similar.…”

Section: Discussionmentioning

confidence: 98%

Comparative analysis of the liver tissue transcriptomes of Mongolian and Lanzhou fat-tailed sheep

Xu¹,

Zhao²,

Yuan³

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Research on gene regulation has been made possible with the help of RNA sequencing applications such as RNA-Seq technology for high-throughput sequencing platforms. Recent studies have explored the transcriptomes from different tissues of domestic animals using RNA-Seq technology, but little research has been done to study the transcriptomes of breeds of sheep having different adipose tissue deposition mechanisms, such as Mongolian and Lanzhou fattailed sheep. In this study, Mongolian and Lanzhou fat-tailed sheep were selected as experimental breeds, and six libraries (three libraries per breed) were constructed. A total of 286 Mb of high-quality reads was obtained, and three-quarters of the reads were mapped to the reference genome per library. In addition, there were 16,257, 16,186, 16,254, 16,827, 16,437, and 15,761 known reference genes in the six constructed libraries (LCL1, LCL2, LCL3, MCL1, MCL2, and MCL3, respectively). Seven genes were differentially expressed: four were upregulated and three were downregulated in liver tissue between the MCL and LCL groups; 65,303, 65,442, 63,426, 76,267, 69,853, and 57,439 potential cSNPs were detected in the six libraries, respectively, with the G/R substitution occurring most commonly. There were 24,239, 22,283, 22,457, 26,635, 27,093, and 18,700 alternate splicing (AS) events in the six libraries. Intron retention was the most common AS event, followed by alternative 3' splice sites. These results indicate that there are many differences in the liver transcriptomes of Mongolian and Lanzhou fat-tailed sheep breeds. Such results may provide fundamental information for further research on defining the sheep genome.

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Section: Discussionmentioning

confidence: 98%

Comparative analysis of the liver tissue transcriptomes of Mongolian and Lanzhou fat-tailed sheep

Xu¹,

Zhao²,

Yuan³

et al. 2016

Genet. Mol. Res.

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“…It was shown that no clear correlation of targeting efficiency was found either with the extent of complementarity between artificial miRNAs and targets or with expression levels of targets 55 . In addition, RNA-seq technique has been shown recently with technical variability particularly in exons at low levels of read coverage, and the relative abundance of a transcript can also substantially disagree 56 . Besides, plant intrinsic variations might contribute to the variations in miRNA expressions, which exhibited as opposite or subtle expression changes compared with the majority, particularly in response to low levels of environmental changes, that is, 26 o C temperature.…”

Section: Discussionmentioning

confidence: 99%

The effects of carbon dioxide and temperature on microRNA expression in Arabidopsis development

et al. 2013

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Elevated levels of CO 2 and temperature can both affect plant growth and development, but the signalling pathways regulating these processes are still obscure. MicroRNAs function to silence gene expression, and environmental stresses can alter their expressions. Here we identify, using the small RNA-sequencing method, microRNAs that change significantly in expression by either doubling the atmospheric CO 2 concentration or by increasing temperature 3-6°C. Notably, nearly all CO 2 -influenced microRNAs are affected inversely by elevated temperature. Using the RNA-sequencing method, we determine strongly correlated expression changes between miR156/157 and miR172, and their target transcription factors under elevated CO 2 concentration. Similar correlations are also found for microRNAs acting in auxin-signalling, stress responses and potential cell wall carbohydrate synthesis. Our results demonstrate that both CO 2 and temperature alter microRNA expression to affect Arabidopsis growth and development, and miR156/157-and miR172-regulated transcriptional network might underlie the onset of early flowering induced by increasing CO 2 .

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“…Illumina sequencing produces negligible technical variability, removing the need for technical replication in RNAseq experiments (Marioni et al, 2008). However, when coverage is low for certain transcripts, technical variation can still arise (McIntyre et al, 2011). Thus, technical replication should be considered for dual RNA-seq experiments where there is low representation of pathogen RNA within a sample, resulting in low coverage of pathogen transcripts.…”

Section: Sample Replicationmentioning

confidence: 99%

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Naidoo¹,

Visser²,

Zwart³

et al. 2018

Current Issues in Molecular Biology

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RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

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RNA-seq: technical variability and sampling

Cited by 269 publications

References 38 publications

Comparative analysis of the liver tissue transcriptomes of Mongolian and Lanzhou fat-tailed sheep

Comparative analysis of the liver tissue transcriptomes of Mongolian and Lanzhou fat-tailed sheep

The effects of carbon dioxide and temperature on microRNA expression in Arabidopsis development

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

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