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Data Analysis

Here are a pair of examples of RNASEQ complete workflows

RNASEQ Pipeline from NCI CCBR

https://github.com/CCBR/Pipeliner/blob/master/RNASeqDocumentation.pdf

RNASEQ Nextflow Pipeline from nf-core

https://nf-co.re/rnaseq

nf_core_rnaseq.pn

Data Quality Assessment (QC)

Basic RNASEQ Quality Control (QC) examines the technical characteristics of the data produced by the sequencer. (It tells us nothing about whether the experiment worked. It answer the questions:

  • Is the data of sufficiently high quality to be analyzed?
  • Are there technical artifacts?

  • Are there poor quality samples?

  • It evaluate the following features

    • Overall sequencing quality scores and distributions
    • GC content distribution
    • Presence of adapter or contamination
  • Sequence duplication levels

Data should be filtered, trimmed, or rejected as appropriate

Sequencing cores generally provide some/all of this analysis

Examples of some of the quantities measured in basic QC

By the program FastQC

https://www.bioinformatics.babraham.ac.uk/projects/fastqc/good_sequence_short_fastqc.html GOOD BAD

By the program MultiQC

https://multiqc.info/examples/rna-seq/multiqc_report.html

Raw Sequence Cleanup

Trim and/or filter sequence to remove sequencing primers/adaptor and poor quality reads. Example programs:

  • FASTX-Toolkit is a collection of command line tools for Short-Reads FASTA/ FASTQ files preprocessing.

  • SeqKit is an ultrafast comprehensive toolkit for FASTA/Q processing.
  • Trimmomatic is a fast, multithreaded command line tool that can be used to trim and crop Illumina (FASTQ) data as well as to remove adapters.
  • TrimGalore is a wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files, with some extra functionality for MspI-digested RRBS-type (Reduced Representation Bisufite-Seq) libraries.
  • Cutadapt finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from your high-throughput sequencing reads.