RNA-Seq-Pipeline - a pipeline to process bulk-RNA-Seq data.

Current Implementation:

• Quality control on raw sequence data (FastQC).
• Revert and merge BAM files (Picard + Samtools).
• Alignment and quantification for both linear and circRNAs (STAR).
• Post-Alignment QC (Picard tools: MarkDuplicates, CollectRNASeqMetrics, CollectAlignmentSummaryMetrics)
• Transcript Integrity (RSeQC, TIN).

Genome Reference & Gene Models (GRCh38)

The genome reference and gene models for the entire pipeline were selected similar to the TOPMed pipeline.

• Reference file:
  • The GRCh38 reference genome and GENCODE 33 annotation, including the addition of ERCC spike-in annotations were used.
  • All ALT, HLA, and Decoy contigs were excluded from the reference genome (cannot be properly handled).
• Reference flat file:
  • The file was generated based on GENCODE 33 + ERCC spike-in annotations for Picard tools (CollectRnaSeqMetrics).
• A ribosomal RNA interval:
  • A list of rRNAs was generated based on GENCODE 33 + ERCC spike-in annotations for Picard tools (CollectRnaSeqMetrics).
• SimpleRepeats & RepeatMasker:
  • repeats were downloaded from UCSC Genome Browser. This file was used with the DCC too for circRNA detection.
• TIN calculations:
  • All protein coding, tag basic, and level 1 transcripts were extracted from the GENCODE 33 + ERCC spike-in annotations.
• Salmon Fasta file:
  • The file was generated based on the reference genome as well as the GENCODE 33 + ERCC spike-in annotations.