Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown

Pertea, Mihaela; Kim, Daehwan; Pertea, Geo; Leek, Jeffrey T.; Salzberg, Steven L.

doi:10.1038/nprot.2016.095

Cited by 4,911 publications

(4,245 citation statements)

References 40 publications

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“…Raw genomic DNA-seq reads were trimmed by Trimmomatic (version 0.33) [61] to remove the universal sequencing adaptors of Illumina with a minimum Phred score of 20 and minimal length of 30 bp. We then mapped the filtered DNA-seq reads using Hisat2 aligner (version 2.0.5) [62] to both sheep reference genome Oar_v4.0 and the alternative genome. Only uniquely aligned reads were kept.…”

Section: Methodsmentioning

confidence: 99%

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Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

et al. 2018

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Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.

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Section: Methodsmentioning

confidence: 99%

“…RNA-seq reads from 15 fetuses (Con: n = 7; Res: n = 4; Over: n = 4) were trimmed and aligned to Oar_v4.0 using Hisat2 version 2.0.5 aligner [62]. The percentages of mapped reads for all samples are summarized in Table S7 and the average multiple aligned rate is 90.3%.…”

Section: Methodsmentioning

confidence: 99%

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

et al. 2018

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“…These were compared to six of the B. germanica libraries: two from fifth instar nymphs, two from sixth instar nymphs and two from adult females. Reads were mapped to the genome using HiSat2 78 . Read counts per gene were obtained using htseq-count and DESeq2 79 was used for differential expression analysis.…”

Section: Nature Ecology and Evolutionmentioning

confidence: 99%

Hemimetabolous genomes reveal molecular basis of termite eusociality

et al. 2018

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E usociality, the reproductive division of labour with overlapping generations and cooperative brood care, is one of the major evolutionary transitions in biology 1 . Although rare, eusociality has been observed in a diverse range of organisms, including shrimps, mole rats and several insect lineages [2][3][4] . A particularly striking case of convergent evolution occurred within the holometabolous Hymenoptera and in the hemimetabolous termites (Isoptera), which are separated by over 350 Myr of evolution 5 . Termites evolved within the cockroaches around 150 Myr ago, towards the end of the Jurassic period 6,7 , about 50 Myr before the first bees and ants appeared 5 . Therefore, identifying the molecular mechanisms common to both origins of eusociality is crucial to understanding the fundamental signatures of these rare evolutionary transitions. While the availability of genomes from many eusocial and non-eusocial hymenopteran species 8 has allowed extensive research into the origins of eusociality within ants and bees [9][10][11] , a paucity of genomic data from cockroaches and termites has precluded large-scale investigations into the evolution of eusociality in this hemimetabolous clade.The conditions under which eusociality arose differ greatly between the two groups. Termites and cockroaches are hemimetabolous and so show a direct development, while holometabolous hymenopterans complete the adult body plan during metamorphosis. In termites, workers are immatures and only reproductive castes are adults 12 , while in Hymenoptera, adult workers and queens represent the primary division of labour. Moreover, termites are diploid and their colonies consist of both male and female workers, and usually a queen and king dominate reproduction. This is in contrast to the haplodiploid system found in Hymenoptera, in which all workers and dominant reproductives are female. It is therefore intriguing that strong similarities have evolved convergently within the termites and the hymenopterans, such as differentiated castes and a nest life with reproductive division of labour. The termites can be subdivided into wood-dwelling and foraging termites. The former belong to the lower termites and produce simple, small colonies with totipotent workers that can become reproductives. Foraging termites (some lower and all higher termites) form large, complex societies, in which worker castes can be irreversible 12 . For this reason, higher, but not lower, termites can be classed as superorganismal 13 . Similarly, within Hymenoptera, varying levels of eusociality exist.Here we provide insights into the molecular signatures of eusociality within the termites. We analysed the genomes of two lower and one higher termite species and compared them to the genome

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“…In preparation for bioinformatic analysis, the RNA-seq reads were processed as follows: reads were trimmed at the 39 end to a length of 65 nt, poly(A) regions were removed, and the read quality was then filtered using the FASTX-Toolkit. The remaining paired-end reads were identified using a custom Python script, and then mapped to the C. albicans SC5314 genome (Assembly 22) using HISAT2 (Kim et al 2015;Pertea et al 2016). The number of reads in genes was counted using the program HTSeqcount (Anders et al 2015).…”

Section: Sequencing Of Complementary Dnas (Cdnas) (Rna-seq)mentioning

confidence: 99%

Regulation of Hyphal Growth and N-Acetylglucosamine Catabolism by Two Transcription Factors in Candida albicans

Naseem

Min

Spitzer³

et al. 2017

Genetics

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The amino sugar N-acetylglucosamine (GlcNAc) is increasingly recognized as an important signaling molecule in addition to its well-known structural roles at the cell surface. In the human fungal pathogen Candida albicans, GlcNAc stimulates several responses including the induction of the genes needed for its catabolism and a switch from budding to filamentous hyphal growth. We identified two genes needed for growth on GlcNAc (RON1 and NGS1) and found that mutants lacking these genes fail to induce the genes needed for GlcNAc catabolism. NGS1 was also important for growth on other sugars, such as maltose, but RON1 appeared to be specific for GlcNAc. Both mutants could grow on nonfermentable carbon sources indicating that they do not affect mitochondrial function, which we show is important for growth on GlcNAc but not for GlcNAc induction of hyphal morphogenesis. Interestingly, both the ron1D and ngs1D mutants were defective in forming hyphae in response to GlcNAc, even though GlcNAc catabolism is not required for induction of hyphal morphogenesis. The ron1D mutant showed a partial defect in forming hyphae, which was surprising since it displayed an elevated level of filamentous cells under noninducing conditions. The ron1D mutant also displayed an elevated basal level of expression of genes that are normally upregulated during hyphal growth. Consistent with this, Ron1 contains an Ndt80-like DNA-binding domain, indicating that it regulates gene expression. Thus, Ron1 is a key new component of the GlcNAc response pathway that acts as both an activator and a repressor of hyphal morphogenesis.

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Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown

Cited by 4,911 publications

References 40 publications

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

Hemimetabolous genomes reveal molecular basis of termite eusociality

Regulation of Hyphal Growth and N-Acetylglucosamine Catabolism by Two Transcription Factors in Candida albicans

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