Using MultiQC

Using MultiQC to produce consolidated QC Reports

Byte Club, October 18 2017
Anna Battenhouse, CSSB & CCBB.

Overview

MultiQC is a tool for aggregating NGS QC reports.
- It does not produce reports, just combines them for unified visualization.
MultiQC "knows" the report formats of many existing NGS tools:
- FastQC, cutadapt, bowtie2, tophat, STAR, kallisto, HISAT2, samtools, featureCounts, HTSeq, MACS2, Picard, GATK
- … and more!
MultiQC can also be configured to display other data via two straightforward steps:

format the data appropriately (e.g. tab-delimited text files)
create appropriate custom data entries in a multiqc_config.yaml configuration file

MultiQC produces neat, interactive plots in an HTML file.
- So it can be used as a basic plotting tool for many kinds of reports and data, not just those produced by NGS tools!

Code Workshop

ATAC-seq is a transposon-insertion sequencing method where an engineered, activate transposon inserts in accessible ("open") chromatin. It is considered to be a much simpler protocol to standard DNase-seq, and requires less starting material as well.

For data, we will use some ATAC-seq datasets produced in Igor Ponomarev's lab in WCAAR. As a proof-of-concept for future work, they performed the ATAC-seq protocol on 5k and 50k cell nuclei from mouse brain, producing 2 paired-end datasets.

Setup to follow along

Login to ls5 at TACC. Execute these commands to set up access to the multiqc binary:

module load python
export PATH="/work/projects/BioITeam/ls5/bin/multiqc-1.0:$PATH"
export PYTHONPATH="/work/projects/BioITeam/ls5/lib/python2.7/annab-packages:$PYTHONPATH"
 
# make sure it is working...
multiqc --help

Produce a consolidated FastQC report

The FastQC took is great for producing detailed reports for every individual fastq file. For example, for Igor's 2 PE datasets, 4 reports are produced from running fastqc (http://web.corral.tacc.utexas.edu/iyer/igor/fastqc/).

The shortcoming is that you have to browse through all the individual reports one at a time, which can be tedious for large experiments.

This is where MultiQC's power comes in. You can point MultiQC to a directory where FastQC has been run and it will magically produce a consolidated report.

For example, logged in to ls5 at TACC, first stage a directory where FastQC has been run:

mkdir -p $SCRATCH/byteclub/multiqc/01_fastq
cd $SCRATCH/byteclub/multiqc/01_fastq
ln -s -f /work/01063/abattenh/projects/byteclub/multiqc/fastqc

Now this is all it takes to produce a basic MultiQC report:

cd $SCRATCH/byteclub/multiqc/01_fastq
multiqc .

When this completes you'll see a new file and directory:

multiqc_report.html – the MultiQC HTML report with its default name
multiqc_data – directory with text files containing MultiQC data used in the report as well as a log file

Here's what this basic FastQC report looks like: http://web.corral.tacc.utexas.edu/iyer/byteclub/multiqc/01_basic.multiqc_report.html

Tip

To view the file you created in a web browser, it must be copied somwhere where a browser can open it. An easy way to do this is to copy it to your laptop like this, for example, changing the user name from abattenh and scratch path as appropriate.

# from your laptop:
scp -p abattenh@ls5.tacc.utexas.edu:/scratch/01063/abattenh/byteclub/multiqc/01_fastq/multiqc_report.html .

Add a few customizations

MultiQC reports can be customized by creating a file called multiqc_config.yaml in the directory where you call multiqc.

Use your favorite text editor to create a a file called multiqc_config.yaml in your $SCRATCH/byteclub/multiqc/01_fastq directory as shown below. This will add report title lines and change the names of the MultiQC output files.

multiqc_config.yaml

# Titles to use for the report.
title: "ATAC-Seq QC Reports"
subtitle: null
intro_text: "MultiQC reports for Igor's ATAC-Seq proof-of-concept project."
report_header_info:
    - Sequenced by: 'GSAF'
    - Job: 'JA17277'
    - Run: 'SA17121'
    - Setup: '2x150'

# Change the output filenames
output_fn_name: mqc_report.html
data_dir_name: mqc_report_data

Catch up

To catch up, just stage Anna's pre-made files:

mkdir -p $SCRATCH/byteclub/multiqc/
cd $SCRATCH/byteclub/multiqc/
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/01_fastq/ 01_fastq/

After saving this file, remove the previous MultiQC outputs and re-run the program:

cd $SCRATCH/byteclub/multiqc/01_fastq
rm -rf multiqc_data multiqc_report.html
multiqc .

If all went well, you should now see a mqc_report.html file and a mqc_report_data directory. Your newly-generated mqc_report.html report file in should look like this (note the new title and header): http://web.corral.tacc.utexas.edu/iyer/byteclub/multiqc/02_custom.mqc_report.html.

Tips for working with the MultiQC configuation file

Here are a few tips for working with the MultiQC configuration file.

Always use spaces (not tabs!) in the multiqc_config.yaml file.
Make sure the file is saved with Unix line endings (not Windows or Mac).
Pay attention to the output when running multiqc. It will tell you if there are issues parsing the config file.
Always delete any previous MultiQC output files before running multiqc
- While their documentation says existing files will just be updated, I have seen MultiQC get confused when previous reports exist.
It is a good idea to change the name of the MultiQC output files
- If output files with those names are not created, something went wrong!
Consult example config files
- An example multiqc_config.yaml file: https ://github.com/ewels/MultiQC/blob/master/multiqc_config_example.yaml
- All multiqc_config.yaml defaults: https://github.com/ewels/MultiQC/blob/master/multiqc/utils/config_defaults.yaml
Avoid running multiqc on large complex directory trees.
- Instead, create a separate directory (or directory tree) only for MultiQC
  - Copy or link the files you want MultiQC to look for there, and use it as MultiQC's target directory.
- MultiQC will run much faster and have fewer confusions.

Add reports from a bowtie2 alignment

First stage some mm10 bowtie2 alignment data:

mkdir -p $SCRATCH/byteclub/multiqc/02_bowtie
cd $SCRATCH/byteclub/multiqc/02_bowtie
ln -s -f /work/01063/abattenh/projects/byteclub/multiqc/fastqc
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/bowtie2/ bowtie2/

Take a look at the contents of the bowtie2 directory. It contains typical output files from running Anna's align_bowtie2_illumina.sh alignment script.

MultiQC will look at all files in this directory looking for report formats it understands. Here, reports that MultiQC will recognize as-is include:

<prefix>.flagstat.txt - output from running samtools flagstat
<prefix>.idxstats.txt - output from running samtools idxstats
<prefix>.dupinfo.txt - output from running Picard MarkDuplicates

Note that output from samtools flagstat and samtools idxstats will only be recognized by MultiQC if the files names include the words flagstat and idxstats. Fortunately, Anna's script created files with those names!

Get ready to re-run MultiQC using the configuration created above.

mkdir -p $SCRATCH/byteclub/multiqc/02_bowtie
cd $SCRATCH/byteclub/multiqc/02_bowtie
cp ../01_fastq/multiqc_config.yaml .

Catch up

To catch up, just use Anna's pre-made files:

mkdir -p $SCRATCH/byteclub/multiqc/
cd $SCRATCH/byteclub/multiqc/
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/03_bowtie/ 02_bowtie/

Now run multiqc again:

cd $SCRATCH/byteclub/multiqc/02_bowtie
rm -rf mqc_report*
multiqc .

If all went well, you should now see a mqc_report.html file that looks like this: http://web.corral.tacc.utexas.edu/iyer/byteclub/multiqc/03_bowtie.mqc_report.html, with new sections for Picard and Samtools reports.

Fix the Picard MarkDuplicates sample name

Notice there is something odd going on in the new General Statistics section. We see M Reads Mapped entries for samples called brain_50k_nuclei and brain_5k_nuclei, but % Dups entries for samples named brain_50k_nuclei.sort and brain_5k_nuclei.sort.

To see where a General Statistics column comes from, hover over the column header. Doing this tells us that the the M Reads Mapped figures came from the samtools flagstat report, while the % Dups comes from Picard MarkDuplicates.

Take a look at one of the <prefix>.dupinfo.txt files to see what might be going on. Below I've added line breaks to the command line info for clarity.

brain_5k_nuclei.dupinfo.txt

## htsjdk.samtools.metrics.StringHeader
# picard.sam.markduplicates.MarkDuplicates INPUT=[brain_5k_nuclei.sort.bam] 
OUTPUT=brain_5k_nuclei.sort.dup.bam 
METRICS_FILE=brain_5k_nuclei.dupinfo.txt ASSUME_SORTED=true VALIDATION_STRINGENCY=LENIENT MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000 MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000 
SORTING_COLLECTION_SIZE_RATIO=0.25 TAG_DUPLICATE_SET_MEMBERS=false 
REMOVE_SEQUENCING_DUPLICATES=false TAGGING_POLICY=DontTag REMOVE_DUPLICATES=false 
DUPLICATE_SCORING_STRATEGY=SUM_OF_BASE_QUALITIES 
PROGRAM_RECORD_ID=MarkDuplicates PROGRAM_GROUP_NAME=MarkDuplicates 
READ_NAME_REGEX=<optimized capture of last three ':' separated fields as numeric values> 
OPTICAL_DUPLICATE_PIXEL_DISTANCE=100 VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 
MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
GA4GH_CLIENT_SECRETS=client_secrets.json
## htsjdk.samtools.metrics.StringHeader
# Started on: Wed Jul 05 23:20:57 CDT 2017

## METRICS CLASS        picard.sam.DuplicationMetrics
LIBRARY UNPAIRED_READS_EXAMINED READ_PAIRS_EXAMINED     SECONDARY_OR_SUPPLEMENTARY_RDS  UNMAPPED_READS       UNPAIRED_READ_DUPLICATES        READ_PAIR_DUPLICATES    READ_PAIR_OPTICAL_DUPLICATES    PERCENT_DUPLICATION  ESTIMATED_LIBRARY_SIZE
brain_5k_nuclei 0       28666117        0       16562322        0       12504025        902024  0.436 195     23118972

## HISTOGRAM    java.lang.Double
BIN     VALUE
1.0     1.016471
...

This is a standard metrics file produced by running Picard MarkDuplicates with a METRICS_FILE option specified. Note the INPUT bam filename: brain_5k_nuclei.sort.bam. MultiQC parses this report looking for the INPUT file, and uses it (minus the .bam suffix) as the sample name. So brain_5k_nuclei.sort.bam becomes sample name brain_5k_nuclei.sort.

The solution is to modify the metrics so that it has brain_5k_nuclei.bam as its INPUT option. This sounds straightforward but there are a couple of gocha's.

We don't want to modify the original metrics file, as it is a faithful record of what was executed. So we want to make a copy of the metrics file with the modified INPUT parameter.
We want MultiQC to ignore the original metrics file, and only use our "fixed" metrics log.
- But MultiQC will, by default, look at all reports in the specified analysis directory (and its sub-directories), and will then find two metrics reports.

For the first part of the solution, we'll create a modified version of the metrics files, but not in the alignment directory, but in a new for_multiqc directory.

mkdir -p $SCRATCH/byteclub/multiqc/02_bowtie/for_multiqc
cd $SCRATCH/byteclub/multiqc/02_bowtie/for_multiqc
for f in ../bowtie2/*.dupinfo.txt; do
  bn=`basename $f`
  pfx=${bn%%.dupinfo.txt}
  echo "$f - $pfx"
  cat $f | sed 's/[.]sort//g' > ${pfx}.dupmetrics.txt
done

Your $SCRATCH/byteclub/multiqc/02_bowtie/for_multiqc directory should have 2 files:

brain_50k_nuclei.fixed.dupmetrics.txt
brain_50k_nuclei.fixed.dupmetrics.txt

The final piece of the puzzle is to tell MultiQC to ignore the original <prefix>.dupinfo.txt files by modifying the multiqc_config.yaml file, adding a fn_ignore_files list entry.

multiqc_config.yaml

# Titles to use for the report.
title: "ATAC-Seq QC Reports"
subtitle: null
intro_text: "MultiQC reports for Igor's ATAC-Seq proof-of-concept project."
report_header_info:
    - Sequenced by: 'GSAF'
    - Job: 'JA17277'
    - Run: 'SA17121'
    - Setup: '2x150'

# Change the output filenames
output_fn_name: mqc_report.html
data_dir_name: mqc_report_data

# Ignore these files / directories / paths when searching for reports
fn_ignore_files:
    - '*.dupinfo.txt'

Catch up

To catch up, just use Anna's pre-made files:

mkdir -p $SCRATCH/byteclub/multiqc
cd $SCRATCH/byteclub/multiqc
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/04_picard_fixed/ 02_bowtie/

After making this config file modification, you can now run multiqc again:

cd $SCRATCH/byteclub/multiqc/02_bowtie; rm -rf mqc_report*; multiqc .

The resulting report should look like this: , with a cleaned up General Statistics table.

Controlling report section order

You may have noticed that MultiQC lists its report sections in fairly random order. If you're Type-A like me, you'll want sections ordered to reflect the sequence of analyses performed. This can be controlled by adding a list of top_module entries.

multiqc_config.yaml

# Titles to use for the report.
title: "ATAC-Seq QC Reports"
subtitle: null
intro_text: "MultiQC reports for Igor's ATAC-Seq proof-of-concept project."
report_header_info:
    - Sequenced by: 'GSAF'
    - Job: 'JA17277'
    - Run: 'SA17121'
    - Setup: '2x150'

# Change the output filenames
output_fn_name: mqc_report.html
data_dir_name: mqc_report_data

# Ignore these files / directories / paths when searching for reports
fn_ignore_files:
    - '*.dupinfo.txt'

# Modules that should come at the top of the report
top_modules:
    - 'generalstats'
    - 'fastqc'
    - 'samtools'
    - 'picard'

Catch up

To catch up, just use Anna's pre-made files:

mkdir -p $SCRATCH/byteclub/multiqc
cd $SCRATCH/byteclub/multiqc
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/05_section_order/ 02_bowtie/

After making this config file modification, you can now run multiqc again:

cd $SCRATCH/byteclub/multiqc/02_bowtie; rm -rf mqc_report*; multiqc .

Producing a report like this: http://web.corral.tacc.utexas.edu/iyer/byteclub/multiqc/05_section_order.mqc_report.html, with a section order that more closely follows workflow processing steps.

About MultiQC custom data

When MultiQC does not know about data produced by a program it doesn't know about, it has a mechanisms for adding custom report sections. The simple way to do this is declaratively, (i.e., via configuation parameters) as described below. (You can also write a Python module for very fine-grained control, but that is a lot more work.)

To add a section for custom data:

Format the data appropriately
- MultiQC supports a number of data file formats (yaml, comma-separated values, etc.)
  - I recommend using simple tab-delimited text files, with MultiQC's preferred .tsv extension.
- data can be provided as one file per sample (where the sample name is part of the file name)
  - or as a single table-like file containing data for all samples
Add two required custom data section entries in the multiqc_config.yaml configuration file
- a sp (search path) section for finding report data
  - specifying a wildcard pattern, if data is supplied as one file per sample
  - or a single file name for a consolidated data file
- each report has a user-named section under a single custom_data section.
  - the required id attribute must be unique, and ties the custom_data, sp and custom_content sections
  - other important attributes include description, file_format, and plot_type.
  - a pconfig sub-section contains plot configuration options
Specify the ordering of the custom report section (optional)
- add a custom_content section order list entry

See http://multiqc.info/docs/#configuration for more details about the structure of custom data sections in multiqc_config.yaml.

MultiQC supports these custom plot types:

bargraph
linegraph
scatter
table
generalstats
beeswarm
heatmap

Plot-type-specific plotting options can be specified in each custom data report's pconfig section. See http://multiqc.info/docs/#plotting-functions for more information. While this section is written for Python programming, the options listed in each plot type's "config" block can be used in the plot's plot's pconfig section (modulo the Python versus YAML formatting differences).

Adding a custom linegraph

Here we'll create a linegraph report of insert sizes for the bowtie2 alignments.

We start with the <prefix>.insertsz.txt files produced by Anna's align_bowtie2_illumina.sh script. That file has both positive and "negative" insert sizes with read counts for all mates, proper pairs, and R1s and R2s individually. For a data file compatible with MultiQC's linegraph, we want only two columns: the positive insert size and count of proper pairs with that size (negative sizes are redundant since each proper pair will have one positive and one negative size entry of the same magnitude).

So a bit of command line reformatting is needed to produce files for MultiQC, which we will save in our for_multiqc directory.

cd $SCRATCH/byteclub/multiqc/02_bowtie/for_multiqc
for f in ../bowtie2/*.insertsz.txt; do
  bn=`basename $f`
  pfx=${bn%%.insertsz.txt}
  echo "$f - $pfx"
  tail -n +2 $f | grep -v -P '^-' | cut -f 1,3 > ${pfx}.bowtie2_isizes.tsv
done

Next we edit the multiqc_config.yaml configuration file to add appropriate custom data sections:

multiqc_config.yaml

# Titles to use for the report.
title: "ATAC-Seq QC Reports"
subtitle: null
intro_text: "MultiQC reports for Igor's ATAC-Seq proof-of-concept project."
report_header_info:
    - Sequenced by: 'GSAF'
    - Job: 'JA17277'
    - Run: 'SA17121'
    - Setup: '2x150'

# Change the output filenames
output_fn_name: mqc_report.html
data_dir_name: mqc_report_data

# Ignore these files / directories / paths when searching for reports
fn_ignore_files:
    - '*.dupinfo.txt'

# Modules that should come at the top of the report
top_modules:
    - 'generalstats'
    - 'fastqc'
    - 'samtools'
    - 'picard'

# --------------------------------
# Custom data
# --------------------------------
custom_content:
  order:
    - bowtie2_isize_section

custom_data:
    bowtie2_isize:
        id: 'bowtie2_isize_section'
        section_name: 'Bowtie2 insert size'
        description: 'distribution for alignments (bowtie2 --local -X2000 --no-mixed --no-discordant)'
        file_format: 'tsv'
        plot_type: 'linegraph'
        pconfig:
            id: 'bowtie2_isize_plot'
            title: 'Insert sizes for proper pairs'
            xlab: 'Insert size'
            ylab: 'Count'

sp:
    bowtie2_isize_section:
        fn: '*.bowtie2_isizes.tsv'

x

Catch up

To catch up, just use Anna's pre-made files:

mkdir -p $SCRATCH/byteclub/multiqc
cd $SCRATCH/byteclub/multiqc
rsync -avrP /work/01063/abattenh/projects/byteclub/multiqc/06_custom_linegraph/ 02_bowtie/

Then the usual...

cd $SCRATCH/byteclub/multiqc/02_bowtie; rm -rf mqc_report*; multiqc .

Resulting in a report that includes our inset size distribution data the custom data section we configured: http://web.corral.tacc.utexas.edu/iyer/byteclub/multiqc/06_custom_linegraph.mqc_report.html, with a new section called Bowtie2 insert size.

What's cool is that this "sawtooth" insert size distribution occurs because of the way transposons insert into the major groove of DNA at regular intervals. So this graph shows Igor that his ATAC-seq proof-of-concept experiment worked!

References

Main MultiQC links

Website: http://multiqc.info/
Documentation: http ://multiqc.info/docs
MultiQC Github repo: https://github.com/ewels/MultiQC
MultiQC test data repo: https://github.com/ewels/MultiQC_TestData

MultiQC configuration files

an example multiqc_config.yaml file: https ://github.com/ewels/MultiQC/blob/master/multiqc_config_example.yaml
all multiqc_config.yaml defaults: https://github.com/ewels/MultiQC/blob/master/multiqc/utils/config_defaults.yaml

MultiQC custom data support

structure of the custom data area of multiqc_config.yaml:
- http://multiqc.info/docs/#configuration
available plot types:
- http://multiqc.info/docs/#plotting-functions
- while this section is written for Python programming, the options listed in each plot type's "config" block can be specified declaratively in any plot's pconfig section in the multiqc_config.yaml.
example custom data files from their test data repo:
- https://github.com/ewels/MultiQC_TestData/tree/master/data/custom_content/no_config
- https://github.com/ewels/MultiQC_TestData/tree/master/data/custom_content/embedded_config

Example Reports from Anna

Below are descriptions of two projects I've assisted with lately using MultiQC to help pull together visualizations assessing experiment quality.

I recommend using Chrome to view MultiQC reports.

The HTML reports generated by MultQC rely heavily on JavaScript and other dynamic web content scripting tools, and not all browsers support them equally well.

These example MultiQC reports below were generated by running the multiqc binary on a command line.
After inspecting them locally (by just opening them as files in a web browser), they were copied to a web-accessible location to share with others. Here, that location is Iyer Lab's web-accessible directory on corral

Igor Ponomarev ATAC-seq data

ATAC-seq is a transposon-insertion sequencing method where an engineered, activate transposon inserts in accessible ("open") chromatin. It is considered to be a much simpler protocol to standard DNase-seq, and requires less starting material as well.

Igor Ponomarev's lab (in WCAAR) performed the ATAC-seq protocol on 5k and 50k cell nuclei from mouse brain, producing 2 paired-end datasets.

http://web.corral.tacc.utexas.edu/iyer/igor/mqc_report.html
has both standard and custom data reports

Marcotte lab amplicon sequencing

The Marcotte lab is working on a deep mutational screening project of a human gene transformed into yeast as an amplicon on a plasmid. Here, the gene is MVK, a gene in the yeast cholesterol biosynthesis pathway. The hsMVK gene is amplified with an error-prone polymerase to produce point mutations. Both the native yeast gene and the human ortholog (with which it shares no sequence similarity) are under on/off promoter control. The idea is to compare the mutations that accumulate in the active hsMVK gene, after many growth cycles, with a background in which the hsMVK gene is present but not active (the yeast MVKis doing the work) to see which mutations are favored or disfavored. As part of this project, Riddhiman Garge produced 19 datasets.

basic FastQC report
- http://web.corral.tacc.utexas.edu/iyer/mvk/mvk_mqc_report.fastqc.html
report on BWA mem alignments of the datasets to hsMVK amplicon and plasmid backbone contigs
- http://web.corral.tacc.utexas.edu/iyer/mvk/mvk_mqc_report.bwa.html
- standard reports from samtools flagstat, samtools idxstats, Picard MarkDuplicates
- custom data reports from bedtools genomecov and from insert size distribution data Anna computed
report using custom data from a specialized deep mutational screening tool from the Jesse Bloom lab
- http://web.corral.tacc.utexas.edu/iyer/mvk/mvk_mqc_report.jbloom.html
- this tool looks only at the overlapping portions of paired-end R1 and R2 reads