Overview of today’s session:
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inspect the results from Session 2
run an advanced RNA-seq pipeline to measure the expression of genes
(optional) run statistical analysis to identify differentially expressed genes
Task 1: Evaluation of RNA-seq results using a basic (generic) nextflow pipeline
The nextflow/RNA-seq pipeline automatically generates two output folders:
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fastqc/ trimgalore/ multiqc/ star_salmon/ pipeline_info/ |
Fastqc FASTQC Report - assessing the quality of input reads
For example; Read1:
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Read 2:
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Connect to the work folder via HPC-FS (See session 2). Browse to the fastqc output folder: run1_star_salmon → results → fastqc. Then click on the HTML reports for each file to assess the quality of raw data. You may also copy the files to your laptop by simply drag-and-drop to a relevant folder.
The main items to verify are denoted below.
Per base sequence quality:
Inspect the overall quality of the generated data per nucleotide position.
Reads with a quality score above 20 (Q20) are 90.0% accurate, and those with >= Q30 are 99.9% accurate.
For most applications, it is recommended to set a quality trimming score of 30. Note, by default the pipeline will remove poor quality reads and bases below Q20.
Per base sequence content:
Determine if biases in the distribution of A, T, C, and G nucleotides are present on either the 5'-end and 3'-end of the reads
Recommendation: remove the first 10 nucleotides from the 5'-end (hexamer primer bias during PCR amplification) and 2 nucleotides from the 3'-end of reads (these bases can interfere with the proper mapping of reads onto reference genomes/transcriptomes).
Check other items reported in the FASTQC report such as level of duplication, highly abundant sequences, and presence of adapter sequences.
MultiQC Report - provides an overview of the quality, trimming, mapping, PCA, and many informative statistics of all files in the experiment in a single report.
Connect to the work folder via HPC-FS (See session 2). Browse to the fastqc output folder: run1_star_salmon → results → multiqc.