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#Next-generation sequencing pipeline Next-generation sequencing methods produce a growing volume of data, leading to increasing difficulties in analysing this data. This manual describes how one can simplify, parallelize and distribute such analysis across high performance compute architecture by using a standardized pipeline and the Molgenis Compute framework.

The pipeline is comprised of best-practice open-source software packages used in multiple institutions leading to 23 analysis steps. The four main parts of the pipeline are:

  • Alignment: here alignment is performed using Burrows-Wheeler Aligner BWA. The produced SAM file is converted to a binary format using Picard and sorted afterwards.
  • Realignment: in this part of the pipeline duplicate reads are marked using Picard. Afterwards realignment around known insertions and deletions (indels) from the Mills-Devine1 dataset using the Genome Analysis ToolKit [GATK] is performed. If reads are re-aligned, the fix-mates step will update the coordinates of the reads mate.
  • Quality score recalibration: Here the base quality scores of a read a recalibrated using covariate information. This method takes several covariates like cycle, dinucleotides, readgroup etc. into account and recalculates the quality scores, leading to reads being re-written with better empirical quality scores.
  • Variant calling: the last part of the pipeline performs indel and SNP calling using the [GATK]. The output of the pipeline are two VCF [reference] files, one with indels and one containing SNPs, ready for downstream analysis.

Expected runtime is about 2 days, depending on whether you are doing exome or whole genome sequencing.

Below is first described how to prepare the 'worksheet' describing your analysis. Then is described how one can execute the analysis pipeline on a cluster (supported backends are Portable Batch System (PBS), Sun Grid Engine (SGE), BSUB or a local machine) and on the national BigGrid Computing infrastructure, eBioGrid.

##Getting started

If you didn't do so, please download compute:

create mycompute #place where you want to work
wget <http address of compute binary>
unzip molgenis-compute-.zip
move molgenis-compute-/* .

All next-generation sequencing protocols are stored in the workflows/ngs/alignmentAndSnpCalling directory. This folder has standard structure of Compute such as parameters.csv, workflow.csv, directory protocols and an example worksheet.csv.

Example data is available in testdata/ngs.

##Preparing the NGS worksheet
Before starting an analysis one has to specify the samples to analyse. For this the worksheet.csv is used. An example worksheet including the obligatory fieds is depicted below:

internalSampleID externalSampleID project sequencingStartDate sequencer run flowcell lane seqType capturingKit barcode barcodeType
autoincrement sample ID projectname date of flowcell sequencer ID run ID flowcell name lane number type of sequencing capturing kit ID barcode used type of barcode

The columns explained:

  • internalSampleID: unique id which is an autoincrement. Type: integer
  • externalSampleID: sample ID. Type: string
  • project: projectname. Type: string
  • sequencingStartDate: date used in the flowcell name in the format of YYYYMMDD. Type: string
  • sequencer: sequencer ID. Type: string
  • run: run number as specified in flowcell name. Type: string
  • flowcell: flowcell name. Type: string
  • lane: lane number, this can be a value between 1-8. Type: int
  • seqType: type of sequencing, either Single Read (SR) or Paired-End (PE). Type: string
  • capturingKit: name of the enrichment kit used, eg. SureSelect_All_Exon_50MB. Type: string
  • barcode: barcode used, eg. CAACCT. Type: string
  • barcodeType: type of barcode used. Type: string

%what are settings for whole genome?

An example worksheet can be found here: workflows/ngs/alignmentAndSnpCalling/worksheet.csv

##Run NGS workflow on cluster or local server Running the pipeline on cluster or local server can be done from the commandline. It consists of the following steps:

  • generate ngs analysis scripts
  • submit ngs analysis scripts
  • monitor

We assume that your are already on the cluster. Otherwise, please move there.

###One time setup of your environement To setup Compute several default parameters in the "parameters.csv" file should be changed to your specific system/cluster environment. Changing these settings is necessary to execute the ngs pipeline. After changing these parameters the parameters file is ready. Changing the following environment parameters is obliged:

  • scheduler: Every scheduler has different job specification syntax, this parameter specifies which header for a specific scheduling system should be generated. The following scheduling systems are supported BSUB (BSUB), Portable Batch System (PBS) and Sun Grid Engine (SGE). To generate jobs for Grid usage the value GRID should be specified.
  • root: this is the "root" parameter shared by all other parameters. To ease the setup we recommend to install all tools in a tools directory and all resources in a resources directory in the "root".

###Generate NGS analysis scripts To generate all jobs for the analysis one can execute the following command (change path to match your worksheet):

sh molgenis_compute.sh \
-workflow=workflows/ngs/alignemntAndSnpCalling/protocols/ \
-worksheet=workflows/ngs/alignemntAndSnpCalling/worksheet.csv \
-outputdir=run01 \
-id=run01

All folders and jobs can be found in the outputdir directory (if ommitted this will be named identical to id).

###Submit NGS analysis scripts The last procedure of the preparation consists of executing the generated shell scripts. For this first copy your analysis to the cluster (if you are not there yet). This can be done by typing the following command (change "run01" to match your outputdir):

cd run01
sh submit.sh

Alternatively, the scripts can be run locally

sh runlocal.sh

Analysis with Compute commandline is now started. A detailed description for execution on the grid can be found in the next chapter.

###Monitor NGS analysis scripts

%todo watch the .finished tags

##Run NGS workflow on BigGrid
Running the pipeline on the grid requires use of MOLGENIS 'pilot' database. This database keeps track of all your jobs (as the grid is distributed and jobs sometimes get lost). Running ngs analysis on the grid consists of the following steps:

###One time setup of your environment If you use BBMRI-NL VO you don't need to install all software binaries. Instead you can use default sofware available via the module system.

% is this needed???

However, you need to setup the path to your data. In parameters.csv change:

  • root: this is the "root" parameter shared by all other parameters. To ease the setup we recommend to install all tools in a tools directory and all resources in a resources directory in the "root".

###Move input data to the grid
Move your data to your $HOME space on the grid and upload it with the "srmcp" command to the "srm" storage. An example command is below (using example data):

upload data to ui node (change path to match your data)

scp testdata/*.fq.gz myname@ui.grid.sara.nl:.

log into ui node (change myname)

ssh myname@ui.grid.sara.nl

put data into srm (change myname and myproject)

mysrm=srm://srm.grid.sara.nl:8443/pnfs/grid.sara.nl/data/bbmri.nl/myname/myproject

srmcp -server_mode=passive file:///$HOME/120308_SN163_0457_BD0E5CACXX_L4_CAACCT_1.fq.gz \
$mysrm/120308_SN163_0457_BD0E5CACXX_L4_CAACCT_1.fq.gz

srmcp -server_mode=passive file:///$HOME/120308_SN163_0457_BD0E5CACXX_L4_CAACCT_2.fq.gz \
$mysrm/120308_SN163_0457_BD0E5CACXX_L4_CAACCT_2.fq.gz

verify

srmls $mysrm

exit

exit

###Start pilot database and load analysis

Start webserver on your own pc (requires external IP, change 8080 if needed)

kill -9 `lsof -i :8080 -t`
cd molgenis_apps;
nohup ant -f build_compute.xml runOn -Dport=8080 &

Import the first workflow into database by running the importWorkflow.sh from deployment directory. Files to be imported can be found here:

sh importWorkflow.sh \
workflows/ngs/alignmentAndSnpCalling/parameters.csv \
workflows/ngs/alignmentAndSnpCalling/workflow.csv \
workflows/ngs/alignmentAndSnpCalling/protocols/

Generate ngs jobs in the database with the importWorksheet.sh from deployment directory and example worksheet:

sh importWorksheet.sh \
workflow.csv \
ui.grid.sara.nl \
workflows/ngs/alignmentAndSnpCalling/worksheet.csv \
test1

###Start pilot jobs on the grid and monitor

Execute ngs analysis with user credentials using pilot job system:

sh runPilots.sh \
ui.grid.sara.nl \
<username> \
<password> \
grid

When you monitor the database at http://localhost:8080 you can see if all your jobs are done (change localhost to remote host if you are running elsewhere).

###Download aligned BAMs & VCF

If so, download your result, when analysis is finished with the same "srmcp" command to UI node

srmcp -server_mode=passive srm://srm.grid.sara.nl:8443/pnfs/grid.sara.nl/ \ data/bbmri.nl/RP2/demo/120308_SN163_0457_BD0E5CACXX_L4_CAACCT.human_g1k_v37.recal.sorted.bam \
file:///$HOME/120308_SN163_0457_BD0E5CACXX_L4_CAACCT.human_g1k_v37.recal.sorted.bam

%can we have an easy script for this???

Footnotes

  1. http://genome.cshlp.org/content/21/6/830.abstract
    [GATK]: http://www.broadinstitute.org/gatk/
    [Molgenis Compute NGS installation manual]: https://github.com/molgenis/molgenis_apps/blob/testing/modules/compute/protocols/ngsWorkflowRealignmentAndSnpCalling/doc/compute_ngs_installation_manual.md