Launching the analysis with STARK

[Nour-EddineS]

Dear @antonylebechec,

I tried to analyze a single BAM file, by the following command: STARK --application=GERMLINE --reads=2336.bam --analysis_name=MyFirstAnalysis --design=target_new.bed --results=STARK/output/results --debug But the order takes more than 22 hours and is not finished yet!! Is this normal ?!. Knowing that my configuration of my station is: CPU: Intel(R) Xeon(R) CPU E5-1620 v3 @ 3.50GHz RAM: 16 GB Distribution: Ubuntu 22.04.1 LTS The output of the command is:

####################################### # STARK # # Stellar Tools for variants # # Analysis and RanKing # # Author: Antony Le Bechec # # Copyright: HUS # # License: GNU GPLA V3 # #######################################

####################################### # Release: 0.9.18.4 # # Date: 20220720 # #######################################

[INFO] Search Application 'GERMLINE'

[INFO] Application 'GERMLINE' found ('/STARK/tools/stark/0.9.18.4/config/apps/GERMLINE.app')

[INFO] Check Samples Analysis

[INFO] Input File '2336.bam' exists

[INFO] Start Samples Analysis

[INFO] /STARK/tools/stark/0.9.18.4/bin/STARK.launch --application=GERMLINE --reads=2336.bam --analysis_name=MyFirstAnalysis --design=target_new.bed --results=STARK/output/results --debug

####################################### # LaunchSample [0.9.7.2-11/11/2021] # Launch a Sample Analysis # Antony Le Bechec @ IRC © GNU-AGPL ####################################### #[DEBUG] F=/STARK/data/2336.bam B=target_new.bed

[DEBUG] FASTQ= /STARK/data/2336.bam

[DEBUG] Format of input file '/STARK/data/2336.bam' ok

[INFO] FOLDER_REPOSITORY=/STARK/output/repository

[INFO] FOLDER_ARCHIVES=/STARK/output/archives

[INFO] FOLDER_FAVORITES=

[INFO] REPOSITORY=

[INFO] ARCHIVES=

[INFO] FAVORITES=

[INFO] REPOSITORY_FILE_PATTERNS= $SAMPLE.tag $SAMPLE..bam.metrics/$SAMPLE..validation.flags.Design.bed $SAMPLE..bam.metrics/$SAMPLE..validation.flags.Panel.bed $SAMPLE..validation.bam $SAMPLE..validation.bam.bai $SAMPLE.reports/$SAMPLE.final.Panel.tsv $SAMPLE.reports/$SAMPLE.final.Panel.vcf.gz $SAMPLE.reports/$SAMPLE.final.Panel.vcf.gz.tbi $SAMPLE.reports/$SAMPLE.final.vcf.gz $SAMPLE.reports/$SAMPLE.final.vcf.gz.tbi $SAMPLE.reports/$SAMPLE.full.Design.tsv $SAMPLE.reports/$SAMPLE.full.Design.vcf.gz $SAMPLE.reports/$SAMPLE.full.Design.vcf.gz.tbi $SAMPLE.reports/..config $SAMPLE.reports/.report.html $SAMPLE.reports/.report.html.folder:FOLDER

[INFO] ARCHIVES_FILE_PATTERNS= $SAMPLE.genes $SAMPLE.tag $SAMPLE.transcripts $SAMPLE..bam.metrics/$SAMPLE..validation.flags..bed $SAMPLE..bam.metrics/$SAMPLE..validation.flags.Panel.bed $SAMPLE.analysis.json $SAMPLE.archive.cram $SAMPLE.archive.cram.crai $SAMPLE.bed $SAMPLE.manifest $SAMPLE.reports/$SAMPLE.final.Panel.vcf.gz $SAMPLE.reports/$SAMPLE.final.Panel.vcf.gz.tbi $SAMPLE.reports/$SAMPLE.final.tsv $SAMPLE.reports/$SAMPLE.final.vcf.gz $SAMPLE.reports/$SAMPLE.final.vcf.gz.tbi $SAMPLE.reports/$SAMPLE.full.vcf.gz $SAMPLE.reports/$SAMPLE.full.vcf.gz.tbi $SAMPLE.reports/..config $SAMPLE.reports/.report.html $SAMPLE.reports/.report.html.folder:FOLDER

[INFO] FAVORITES_FILE_PATTERNS=

FASTQ /STARK/data/2336.bam FASTQ_R2 SAMPLE 2336 RUN MyFirstAnalysis BED target_new.bed GENES TRANSCRIPTS PEDIGREE INPUT /STARK/data OUTPUT RESULTS STARK/output/results PIPELINES bwamem.gatkHC_GERMLINE.howard bwamem.gatkUG_GERMLINE.howard

[INFO] *** Start Analysis [Tue Jan 31 10:56:13 UTC 2023]

[INFO] *** Input

[INFO] Check Input Files...

[INFO] RUN 'MyFirstAnalysis'

[DEBUG] /STARK/data/2336.bam | | | | | 2336 | MyFirstAnalysis | target_new.bed | | | | bwamem.gatkHC_GERMLINE.howard bwamem.gatkUG_GERMLINE.howard | /STARK/data | STARK/output/results | STARK/output/results/MyFirstAnalysis/2336

[INFO] RUN 'MyFirstAnalysis' - SAMPLE '2336'

[INFO] SAMPLE 'MyFirstAnalysis/2336' from file(s):

[INFO] Read1: /STARK/data/2336.bam

[INFO] Read2:

[INFO] Index1:

[INFO] Index2:

[INFO] Others:

[INFO] Design: target_new.bed

[INFO] Panels:

[INFO] Transcripts:

[INFO] Pedigree:

[INFO] Tags:

[INFO] Create Input data from BAM/CRAM/SAM file

[INFO] FASTQ processing (Adaptors, UMIs, quality)

[INFO] Copy original BED file.

[INFO] Sort/Merge/Normalize BED file.

[INFO] Create LIST.GENES file 'STARK/output/results/MyFirstAnalysis/2336/2336.list.genes' from Design file 'target_new.bed'.

[INFO] Create LIST.GENES file 'STARK/output/results/MyFirstAnalysis/2336/2336.list.genes' with intersection between Design file 'target_new.bed' and RefSeq '/STARK/databases/refGene/current/refGene.hg19.bed'.

[INFO] Generate genes.bed from .genes files within LIST.GENES file 'STARK/output/results/MyFirstAnalysis/2336/2336.list.genes'.

[INFO] Create TAG file.

[INFO] Create Analysis TAG file.

[INFO] Copy SampleSheet.

[INFO] *** Configuration

[INFO] * ANALYSIS

[INFO] ANALYSIS NAME MyFirstAnalysis

[INFO] ANALYSIS TAG

[INFO] * SAMPLES

[INFO] SAMPLE NAMES 2336

[INFO] SAMPLE TAG

[INFO] FASTQ/BAM/CRAM /STARK/data/2336.bam

[INFO] FASTQ R2

[INFO] INDEX1

[INFO] INDEX2

[INFO] OTHER_FILES

[INFO] DESIGN target_new.bed

[INFO] GENES STARK/output/results/MyFirstAnalysis/2336/2336.from_design.genes

[INFO] TRANSCRIPTS

[INFO] PEDIGREE

[INFO] * APPLICATION

[INFO] APPLICATION NAME GERMLINE:1.0

[INFO] APPLICATION FILE GERMLINE.app

[INFO] GROUP GENETIC

[INFO] PROJECT GERMLINE

[INFO] PIPELINES bwamem.gatkHC_GERMLINE.howard bwamem.gatkUG_GERMLINE.howard

[INFO] POST SEQUENCING

[INFO] POST ALIGNMENT sorting markduplicates realignment recalibration compress

[INFO] POST CALLING

[INFO] POST ANNOTATION

[INFO] RESULTS STARK/output/results

[INFO] REPOSITORY

[INFO] ARCHIVES

[INFO] FAVORITES

[INFO] RELEASE INFOS STARK/output/results/MyFirstAnalysis/STARK.20230131-105613.analysis.release

[INFO] MAKEFILE CONFIGURATION STARK/output/results/MyFirstAnalysis/STARK.20230131-105613.analysis.param.mk

[INFO] LOGFILE STARK/output/results/MyFirstAnalysis/STARK.20230131-105613.analysis.log

[INFO] THREADS 7

[INFO] THREADS_BY_SAMPLE 7

[INFO] THREADS_COPY 1

[INFO] *** Process

[INFO] STARK Input Processing...

[INFO] Process Input data from FASTQ file(s) - multithreading mode [7]

[INFO] STARK Input Processing done.

[INFO] STARK Analysis Processing...

Best regards,

[antonylebechec]

Dear @Nour-EddineS,

First of all, your command seems to be healthy.

Then, the execution time of your command depends on the input data (e.g. BAM, BED) and resources (e.g. CPU, MEM, network, disk).

About input data, can you provide:

• BAM size (on disk) and stats (nb of reads, average depth...)
• BED size (short panel? large panel? exome?)

About resources:

• We recommend to use at least 8Go per CPU. I've noticed that you are using 8 threads (4 cores with 2 threads each) and 16Go of RAM. By default, STARK calculates the number of threads to use (total number minus 1, to keep the system available on 1 thread at least). So, your analysis uses 7 threads and 16Go of RAM, which is not the correct ratio (1CPU/8Go). This is particularly important if your analysis takes into account several samples (which is not the case here).

Furthermore, the GERMLINE application is configured with 1 aligner (bwamem) and 2 callers (gatkHC_GERMLINE and gatkUG_GERMLINE). Especially GATKUG (Unified Genotyper) takes a while and is deprecated. I suggest you use the default application which is configured with only 1 caller (gatkHC, Haplotype Caller). You can also configure your own application if needed.

Also remember that STARK generates metrics, annotations, reports and other information that are useful for data interpretation, but will increase execution time, compared to a simple pipeline (alignment and calling).

Finally, to check if your analysis is really healthy, can you have a look at the processes and generated data?

• htop -t
• in $HOME/STARK/output/results/MyFirstAnalysis, check .bam, .vcf.gz files, as well as the main log file (analysis.log) and the configuration file (config).

Hope this helps!

Best regards,