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NGI-MethylSeq Output

NGI-MethylSeq is the new RNA-seq Best Practice pipeline used by the National Genomics Infrastructure at SciLifeLab in Stockholm, Sweden.

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

Note that NGI-MethylSeq contains two workflows - one for Bismark, one for bwa-meth. This document describes the output from the default Bismark workflow.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

FastQC

FastQC gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, the per base sequence content (%T/A/G/C). You get information about adapter contamination and other overrepresented sequences.

For further reading and documentation see the FastQC help.

NB: The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequence and potentially regions with low quality. To see how your reads look after trimming, look at the FastQC reports in the trim_galore directory.

Output directory: results/fastqc

  • sample_fastqc.html
    • FastQC report, containing quality metrics for your untrimmed raw fastq files
  • sample_fastqc.zip
    • zip file containing the FastQC report, tab-delimited data file and plot images

TrimGalore

The NGI-MethylSeq BP 2.0 pipeline uses TrimGalore for removal of adapter contamination and trimming of low quality regions. TrimGalore uses Cutadapt for adapter trimming and runs FastQC after it finishes.

MultiQC reports the percentage of bases removed by TrimGalore in the General Statistics table, along with a line plot showing where reads were trimmed.

Output directory: results/trim_galore

Contains FastQ files with quality and adapter trimmed reads for each sample, along with a log file describing the trimming.

  • sample_val_1.fq.gz, sample_val_2.fq.gz
    • Trimmed FastQ data, reads 1 and 2.
    • NB: Only saved if --saveTrimmed has been specified.
  • logs/sample_val_1.fq.gz_trimming_report.txt
    • Trimming report (describes which parameters that were used)
  • FastQC/sample_val_1_fastqc.zip
    • FastQC report for trimmed reads

Single-end data will have slightly different file names and only one FastQ file per sample.

Bismark

The default NGI-MethylSeq workflow uses Bismark to process raw sequencing reads into cytosine methylation calls.

Alignment

Bismark converts all Cytosines to Thymine in-silico and then aligns against a three-letter reference genome. It produces a BAM file of genomic alignments.

Output directory: results/bismark_alignments/

  • sample.bam
    • Aligned reads in BAM format.
    • Only saved if --saveAlignedIntermediates, --nodedup or --rrbs is specified when running the pipeline.
  • logs/sample_PE_report.txt
    • Log file giving summary statistics about alignment.
  • unmapped/unmapped_reads_1.fq.gz, unmapped/unmapped_reads_2.fq.gz
    • Unmapped reads in FastQ format.
    • Only saved if --unmapped specified when running the pipeline.

Deduplication

This step removes alignments with identical mapping position to avoid technical duplication in the results. Note that it is skipped if --saveAlignedIntermediates, --nodedup or --rrbs is specified when running the pipeline.

Output directory: results/bismark_deduplicated/

  • deduplicated.bam
    • BAM file with only unique alignments.
  • logs/deduplication_report.txt
    • Log file giving summary statistics about deduplication.

Methylation Extraction

The bismark methylation extractor tool takes a BAM file aligned with Bismark and generates files containing methylation information about cytosines. It produces a few different output formats, described below.

Note that the output may vary a little depending on whether you specify --comprehensive or --non_directional when running the pipeline.

Filename abbreviations stand for the following reference alignment strands:

  • OT - original top strand
  • OB - original bottom strand
  • CTOT - complementary to original top strand
  • CTOB - complementary to original bottom strand

(CTOT and CTOB are not aligned unless --non_directional specified).

Output directory: results/bismark_methylation_calls/

  • methylation_calls/XXX_context_sample.txt.gz
    • Individual methylation calls, sorted into files according to cytosine context.
  • methylation_coverage/sample.bismark.cov.gz
    • Coverage text file summarising cytosine methylation values.
  • bedGraph/sample.bedGraph.gz
    • Methylation statuses in bedGraph format, with 0-based genomic start and 1- based end coordinates.
  • m-bias/sample.M-bias.txt
    • QC data showing methylation bias across read lengths. See the bismark documentation for more information.
  • logs/sample_splitting_report.txt
    • Log file giving summary statistics about methylation extraction.

Report

Bismark generates a HTML report describing results for each sample.

Output directory: results/bismark_reports

Summary

Bismark generates summary text and HTML reports giving an overview of results for all samples in a project.

Output directory: results/bismark_summary

Qualimap

Qualimap BamQC is a general-use quality-control tool that generates a number of statistics about aligned BAM files. It's not specific to bisulfite data, but it produces several useful stats - for example, insert size and coverage statistics.

Output directory: results/qualimap

  • sample/qualimapReport.html
    • Qualimap HTML report
  • sample/genome_results.txt, sample/raw_data_qualimapReport/*.txt
    • Text-based statistics that can be loaded into downstream programs

MultiQC

MultiQC is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in within the report data directory.

Output directory: results/multiqc

  • Project_multiqc_report.html
    • MultiQC report - a standalone HTML file that can be viewed in your web browser
  • Project_multiqc_data/
    • Directory containing parsed statistics from the different tools used in the pipeline

For more information about how to use MultiQC reports, see http://multiqc.info

SciLifeLab National Genomics Infrastructure