Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken

Abstract

BackgroundThe domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues.ResultsRandomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development.ConclusionExpression profiles obtained from public RNA-seq datasets – despite being generated by different laboratories using different methodologies – can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4972-7) contains supplementary material, which is available to authorized users.

Functional Annotation of the Transcriptome of the Pig, Sus scrofa, Based Upon Network Analysis of an RNAseq Transcriptional Atlas

“…We created an expression atlas for the domestic pig by aggregating publicly archived RNAseq libraries, using a pipeline previously described for chickens (Bush et al, 2018). To identify candidate libraries, we downloaded the daily updated SRA BioProject summary file (n = 355,400 BioProjects; ftp://ftp.…”

“…For each library, expression was quantified using Kallisto v0.44.0 (Bray et al, 2016) as described in detail in previous studies on other species (Clark et al, 2017;Bush et al, 2018;Young et al, 2019). Kallisto quantifies expression at the transcript level, as transcripts per million (TPM), by building an index of k-mers from a set of reference transcripts and then "pseudo-aligning" reads to it, matching k-mers in the reads to k-mers in the index.…”

“…The trimmed UTRs from each mRNA were generated excluding all sequence outside the longest ORF. The method of integrating transcripts from both Ensembl and NCBI data sets is as previously described (Bush et al, 2018).…”

“…As a final quality control test (described in (Bush et al, 2018)) we considered that in a correctly prepared RNAseq library, a minority of genes will produce the majority of reads and so the distribution of gene-level TPM estimates should comply, to a reasonable approximation, with Zipf's law (i.e., that the probability of an observation is inversely proportional to its rank). On this basis, the exponent of a log-log plot of the reverse cumulative TPM per gene should not substantially deviate from an optimal value of −1.…”

“…There are numerous additional pig RNAseq data sets from diverse tissues, breeds, and conditions in the public domain. We recently developed an approach to harvest and normalize such published data to create an expression atlas for the chicken (Bush et al, 2018). Based upon the use of the high-speed "pseudoaligner" Kallisto (Bray et al, 2016) to quantify expression this atlas was rapidly created and easily scalable to include new data sets.…”