Multi-perspective quality control of Illumina RNA sequencing data analysis

Abstract

Quality control (QC) is a critical step in RNA sequencing (RNA-seq). Yet, it is often ignored or conducted on a limited basis. Here, we present a multi-perspective strategy for QC of RNA-seq experiments. The QC of RNA-seq can be divided into four related stages: (1) RNA quality, (2) raw read data (FASTQ), (3) alignment and (4) gene expression. We illustrate the importance of conducting QC at each stage of an RNA-seq experiment and demonstrate our recommended RNA-seq QC strategy. Furthermore, we discuss the major and often neglected quality issues associated with the three major types of RNA-seq: mRNA, total RNA and small RNA. This RNA-seq QC overview provides comprehensive guidance for researchers who conduct RNA-seq experiments.

The Landscape of Small Non-Coding RNAs in Triple-Negative Breast Cancer

“…Cutadapt [ 23 ] was used to trim 3′ adapters for raw reads. Multi-perspective quality control [ 24 ] on raw data was performed using QC3 [ 25 ]. All reads with length less than 16 nucleotides were discarded.…”

Moving beyond neurons: the role of cell type-specific gene regulation in Parkinson’s disease heritability

“…We thereafter selected the top 10% of genes enriched within each cell type and added a 100kb window to reflect the approach used by Finucane et al 17 When using the Barres data, we averaged gene expression across samples of the same cell type, filtered genes on the basis of an FPKM ≥ 1 in at least one cell type (this equates to ~66% of all genes with FPKM > 0.1, which was set by Zhang et al 20 as the threshold for minimum gene expression), and then calculated gene enrichment. Our detection threshold of FPKM ≥ 1 was employed on the basis that smaller thresholds tend to produce large and misleading enrichments 51 . The Linnarsson data was available with gene expression aggregated by sub-cell type/cluster.…”

Expression correlation attenuates within and between key signaling pathways in chronic kidney disease

“…Total RNA extraction with ribosomal RNA removal (Ribo-Zero), RNA quality control, strand-specific library construction (Illumina), and 150 bp pair end RNA sequencing (Illumina) were conducted by Novogene. RNA-Seq data were quality controlled according to multi-perspective guideline [ 23 ] using QC3 [ 24 ]. Alignments were performed using STAR [ 25 ] against the GRCh38 reference genome, gene quantification was done using Cufflinks [ 26 ].…”