Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.

2019-06-27 | GSE131397 | GEO

Strand-specific Illumina TruSeq mRNA sequencing libraries of 1000 Genomes Trios

Project description:Strand-specific Illumina TruSeq mRNA sequencing libraries of 1000 Genomes Trios

| PRJEB11271 | ENA

Amplicon-based targeted re-sequencing analysis of target genes in human DNA samples using the Illumina TruSeq Custom Amplicon technology

Project description:Amplicon-based targeted re-sequencing analysis was performed in the patient-derived gliobastoma cell culture samples. For this purpose, genomic DNA (gDNA) was isolated and DNA libraries were prepared using the TruSeq Custom Amplicon Low Input (Illumina, Inc.) technology. By this, a pool of 375 amplicons was generated for each single sample in order to enrich for the target genes ATRX1, EGFR, IDH1, NF1, PDGFRA, PIK3CG, PIK3R1, PTEN, RB1 and TP53. Sequencing was performed on the Illumina MiSeq® next generation sequencing system (Illumina Inc.) and its 2 x 250 bp paired-end v2 read chemistry. The resulting reads were quality controlled and mapped against the human reference genome (hg19). For all samples, sequence variations of the amplified regions of interest in comparison to the human reference sequence were identified and filtered based on reliability.

2021-07-07 | E-MTAB-9343 | biostudies-arrayexpress

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

Project description:BackgroundRibosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples.ResultsWe find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency.ConclusionsThese results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

| S-EPMC6389247 | biostudies-literature

Fractionation RNAseq in conjunction with Illumina TRUseq method

Project description:H9 human ESCs (H9 line) were cultured in BMP4 cultured medium on Matrigel-coated plates. Colonies were passaged for maintanence by Gentle Cell Dissociation Reagent

2019-03-20 | E-MTAB-5116 | biostudies-arrayexpress

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