Scientific IT Services Michal - PowerPoint Presentation

Slide1

Scientific IT ServicesMichal Okoniewski, Samuel Fux, Manuel Kohler

11/22/17

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High Performance Computing for genomic applicationsUsing genomic software on Euler

Michal Okoniewski, Scientific IT ETH

module loadmodule load gdcmodule avail

module purge

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Michal Okoniewski, Scientific IT ETH 2Bioinformatic modules on EULER

Data processing and convertion: samtools, picard,

…Aligners: bwa,

bowtie, SHRiMP, …

RNA aligners: STAR, tophat, subjuncTranscriptome aligners: kallistio

, sailfish, RSEM, …

Old

-style

aligners

: Blast, Blat, VMATCH

De-

novo

assemblers

: trinity, velvet, spades,…Feature extraction, counting: HTSeq, featureCountTranscript discovery: cufflinksSpecialized tools: MISO, blast2go…

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Michal Okoniewski, Scientific IT ETH

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Bioinformatic

software jungle - categories

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Bioinformatic software jungle

other

MISO/MATS

jsplice

cufflinks/

cuffdiff

SparkSeq

tests

DESeq

/

edgeR

DEXSeq

BAM

BAM

BAM

BAM

fastq

fastq

fastq

fastq

STAR

aligner

fastqc

Fastqc

report

ht_seq

SparkSeq

counts

SparkSeq

junctions

junctions

junctions

junctions

junctions

RPKM

tables

Count table

Count tables

(gene, exon…)

RPKM

tables

RSEM/

Bitseq

isoform

deconvolute

tophat

aligner

Experiment

definition

REPORTING PANEL

Selection of

graphs

Report types

output formats (BED CSV..)

Setting thresholds

Functional analysis

parametres

David

String DB

GeneGo

(commercial)

Ingenuity

(commercial)

Differential expression

report

Differential splicing

report

fastq

Functional analysis

tools

Statistical

analysis

tools

Genomic feature

extraction

- counting

Alignment

to the genome

CSV

BED

Reporting

Output

Genome

browser

Genome

browser

Genome

browser

Filters,

trimming

Filters,

trimming

unmapped

fastq

unmapped

fastq

unmapped

BAM

unmappedBAM

igenomes

GTF/GFF

genome

STARTING

PANEL

OptionsFor analysisMode:Step-by-stepSingle-run

Building genome indexbowtie2-build

--threads 24 /

cluster/home/michalo

/work_michalo/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa /cluster/scratch/michalo/hg38/hg38

Alignment:

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Michal Okoniewski, Scientific IT ETH

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Bowtie, bowtie2

Classic splice-aware alignerUses bowtie2 as engine, so also bowtie2 index t

ophat -p

24 -o tophat_out

--library-type fr-firststrand ~/work_michalo

/hg38/hg38 mini.fastq.gz

Manual: http://

ccb.jhu.edu

/software/

tophat

/

index.shtml

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Michal Okoniewski, Scientific IT ETH

6Tophat

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“Tuxedo suite”

Transcript discovery toolUses coverage and junctions from a BAM file

cufflinks mini_star.sorted.bam

Other cuffmerge

, cuffdiff, cuffquant, cuffnorm, CummeRbund11/22/17

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Cufflinks

Produces GTF

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9Cufflinks

Splice aware aligner, loading index into memoryResults similar to tophat, but faster--genomeLoad

LoadAndKeepWith specific options, can produce BAM and do the counting too

https://github.com/alexdobin/STAR/blob/master/doc/

STARmanual.pdf11/22/17Michal Okoniewski, Scientific IT ETH

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STAR

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STAR

Includes subjunc similar to STAR and featureCounts Building index

subread-buildindex -o /cluster/home/

michalo/

work_michalo/hg38/subread_index/hg38 /

cluster/home/michalo

/

work_michalo

/hg38/Homo_sapiens.GRCh38.dna.primary_assembly.fa

Alignment

subread

-T 24 -

i

/cluster/home/michalo/work_michalo/hg38/subread_index/hg38 -r

mini.fastq

-o mapped_reads_subjunc

/mini.bam

subjunc

-T 24 -

i

/cluster/home/

michalo

/

work_michalo

/hg38/

subread_index

/hg38 -r

mini.fastq

-o

mapped_reads_subjunc

/

mini.bam

http

://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf

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subread

General purpose tool for conversion of BAM  SAM Many other operations: pileup…

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13samtools

Fast and flexible counting in genomic featuresfeatureCounts

-M -s 2 -T 24 -t gene -g gene_id

-a /cluster/home/michalo

/work_michalo/hg38/Homo_sapiens.GRCh38.86.chr.gtf -o mini.cnt

mini_star.sorted.bam

Important options:

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featureCounts

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featureCounts

GATK is a genomic toolbox for various operations related mainly to genomic variants calling Operations include producing a variant file *.vcf

from an alignment file *.bam module load

gcc/4.8.2

gdc java/1.8.0_73 gatk/3.5java

-jar GenomeAnalysisTK.jar

-T

UnifiedGenotyper

-

R ref/human_g1k_b37_20.fasta

-

I

bams

/exp_design/NA12878_wgs_20.bam -o sandbox/NA12878_wgs_20_UG_calls.vcf -

glm BOTH

-L

20:10,000,000-10,200,000https://software.broadinstitute.org/gatk/documentation/tooldocs/current/

https://software.broadinstitute.org/gatk/documentation/topic?name=tutorials

http://gatkforums.broadinstitute.org/gatk/discussion/7869/howto-discover-variants-with-gatk-a-gatk-workshop-tutorial

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GATK

Using

genomic software on Euler

Thank you!