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The**scope**of**this**OMICS**MASTER**solution**is**restricted**to**human**genomics**research**(disease**causing**gene**discovery**in**whole**human**genome**or**exome)**or**diagnosis**(panel**sequencing),**although**it**could**be**extended**in**the**future**to**other**usages.
The**pipeline**inputs**the**raw**data**produced**by**the**sequencing**machines**and**undergoes**a**processing**procedure**that**consists**on**a**quality**control,**the**mapping**and**variant**calling**steps**that**result**in**a**file**containing**the**set**of**variants**in**the**sample.**From**this**point,**the**prioritization**component**or**the**diagnostic**component**can**be**launched.
**Figure**1.**OMICS**MASTER**solution**overview.**Data**is**produced**in**the**external**labs**and**comes**to**IT4I**(represented**by**the**blue**dashed**line).**The**data**pre-processor**converts**raw**data**into**a**list**of**variants**and**annotations**for**each**sequenced**patient.**These**lists**files**together**with**primary**and**secondary**(alignment)**data**files**are**stored**in**IT4I**sequence**DB**and**uploaded**to**the**discovery**(candidate**prioritization)**or**diagnostic**component**where**they**can**be**analyzed**directly**by**the**user**that**produced**them,**depending**of**the**experimental**design**carried**out.
Typical**genomics**pipelines**are**composed**by**several**components**that**need**to**be**launched**manually.**The**advantage**of**OMICS**MASTER**pipeline**is**that**all**these**components**are**invoked**sequentially**in**an**automated**way.
OMICS**MASTER**pipeline**inputs**a**FASTQ**file**and**outputs**an**enriched**VCF**file.**This**pipeline**is**able**to**queue**all**the**jobs**to**PBS**by**only**launching**a**process**taking**all**the**necessary**input**files**and**creates**the**intermediate**and**final**folders
Let’s**see**each**of**the**OMICS**MASTER**solution**components:
This**component**is**composed**by**a**set**of**programs**that**carry**out**quality**controls,**alignment,**realignment,**variant**calling**and**variant**annotation.**It**turns**raw**data**from**the**sequencing**machine**into**files**containing**lists**of**variants**(VCF)**that**once**annotated,**can**be**used**by**the**following**components**(discovery**and**diagnosis).
We**distinguish**three**types**of**sequencing**instruments:**bench**sequencers**(MySeq,**IonTorrent,**and**Roche**Junior,**although**this**last**one**is**about**being**discontinued),**which**produce**relatively**Genomes**in**the**clinic
low**throughput**(tens**of**million**reads),**and**high**end**sequencers,**which**produce**high**throughput**(hundreds**of**million**reads)**among**which**we**have**Illumina**HiSeq**2000**(and**new**models)**and**SOLiD.**All**of**them**but**SOLiD**produce**data**in**sequence**format.**SOLiD**produces**data**in**a**special**format**called**colour**space**that**require**of**specific**software**for**the**mapping**process.**Once**the**mapping**has**been**done,**the**rest**of**the**pipeline**is**identical.**Anyway,**SOLiD**is**a**technology**which**is**also**about**being**discontinued**by**the**manufacturer**so,**this**type**of**data**will**be**scarce**in**the**future.
####**Quality**Control,**Preprocessing**and**Statistics**for**FASTQ
These**steps**are**carried**out**over**the**original**FASTQ**file**with**optimized**scripts**and**includes**the**following**steps:**sequence**cleansing,**estimation**of**base**quality**scores,**elimination**of**duplicates**and**statistics.
Output:****FASTQ**file**plus**an**HTML**file**containing**statistics**on**the**data**.
FASTQ**format**It**represents**the**nucleotide**sequence**and**its**corresponding**quality**scores.
![FASTQ**file.](../../../img/fig2.png**"fig2.png")
**Figure**2**.FASTQ**file.
Sequence**reads**are**mapped**over**the**human**reference**genome.**SOLiD**reads**are**not**covered**by**this**solution;**they**should**be**mapped**with**specific**software**(among**the**few**available**options,**SHRiMP**seems**to**be**the**best**one).**For**the**rest**of**NGS**machine**outputs**we**use**HPG**Aligner.**HPG-Aligner**is**an**innovative**solution,**based**on**a**combination**of**mapping**with**BWT**and**local**alignment**with**Smith-Waterman**(SW),**that**drastically**increases**mapping**accuracy**(97%**versus**62-70%**by**current**mappers,**in**the**most**common**scenarios).**This**proposal**provides**a**simple**and**fast**solution**that**maps**almost**all**the**reads,**even**those**containing**a**high**number**of**mismatches**or**indels.
It**is**a**human**readable**tab-delimited**format**in**which**each**read**and**its**alignment**is**represented**on**a**single**line.**The**format**can**represent**unmapped**reads,**reads**that**are**mapped**to**unique**locations,**and**reads**that**are**mapped**to**multiple**locations.
The**SAM**format**(1)**consists**of**one**header**section**and**one**alignment**section.**The**lines**in**the**header**section**start**with**character**‘@’,**and**lines**in**the**alignment**section**do**not.**All**lines**are**TAB**delimited.
In**SAM,**each**alignment**line**has**11**mandatory**fields**and**a**variable**number**of**optional**fields.**The**mandatory**fields**are**briefly**described**in**Table**1.**They**must**be**present**but**their**value**can**be**a**‘\*’**or**a**zero**(depending**on**the**field)**if**the
corresponding**information**is**unavailable.
|****No.****|****Name****|****Description****************************************************************************|
|**---------**|**----------**|**-----------------------------------------------------**|
|**1******************|**QNAME************|**Query**NAME**of**the**read**or**the**read**pai********************************|
|**2******************|**FLAG**************|**Bitwise**FLAG**(pairing,strand,mate**strand,etc.)****************|
|**3******************|**RNAME************|**<p>Reference**sequence**NAME********************************************************|
|**4******************|**POS****************|**<p>1-Based**leftmost**POSition**of**clipped**alignment********|
|**5******************|**MAPQ**************|**<p>MAPping**Quality**(Phred-scaled)******************************************|
|**6******************|**CIGAR************|**<p>Extended**CIGAR**string**(operations:MIDNSHP)******************|
|**7******************|**MRNM**************|**<p>Mate**REference**NaMe**('='**if**same**RNAME)************************|
|**8******************|**MPOS**************|**<p>1-Based**leftmost**Mate**POSition******************************************|
|**9******************|**ISIZE************|**<p>Inferred**Insert**SIZE**************************************************************|
|**10****************|**SEQ****************|**<p>Query**SEQuence**on**the**same**strand**as**the**reference**|
|**11****************|**QUAL**************|**<p>Query**QUALity**(ASCII-33=Phred**base**quality)****************|
The**standard**CIGAR**description**of**pairwise**alignment**defines**three**operations:**‘M’**for**match/mismatch,**‘I’**for**insertion**compared**with**the**reference**and**‘D’**for**deletion.**The**extended**CIGAR**proposed**in**SAM**added**four**more**operations:**‘N’**for**skipped**bases**on**the**reference,**‘S’**for**soft**clipping,**‘H’**for**hard**clipping**and**‘P’**for**padding.**These**support**splicing,**clipping,**multi-part**and**padded**alignments.**Figure**3**shows**examples**of**CIGAR**strings**for**different**types**of**alignments.
**Figure**3****.**SAM**format**file.**The**‘@SQ’**line**in**the**header**section**gives**the**order**of**reference**sequences.**Notably,**r001**is**the**name**of**a**read**pair.**According**to**FLAG**163**(=1+2+32+128),**the**read**mapped**to**position**7**is**the**second**read**in**the**pair**(128)**and**regarded**as**properly**paired**(1**+**2);**its**mate**is**mapped**to**37**on**the**reverse**strand**(32).**Read**r002**has**three**soft-clipped**(unaligned)**bases.**The**coordinate**shown**in**SAM**is**the**position**of**the**first**aligned**base.**The**CIGAR**string**for**this**alignment**contains**a**P**(padding)**operation**which**correctly**aligns**the**inserted**sequences.**Padding**operations**can**be**absent**when**an**aligner**does**not**support**multiple**sequence**alignment.**The**last**six**bases**of**read**r003**map**to**position**9,**and**the**first**five**to**position**29**on**the**reverse**strand.**The**hard**clipping**operation**H**indicates**that**the**clipped**sequence**is**not**present**in**the**sequence**field.**The**NM**tag**gives**the**number**of**mismatches.**Read**r004**is**aligned**across**an**intron,**indicated**by**the**N**operation.
BAM**is**the**binary**representation**of**SAM**and**keeps**exactly**the**same**information**as**SAM.**BAM**uses**lossless**compression**to**reduce**the**size**of**the**data**by**about**75%**and**provides**an**indexing**system**that**allows**reads**that**overlap**a**region**of**the**genome**to**be**retrieved**and**rapidly**traversed.
####**Quality**Control,**Preprocessing**and**Statistics**for**BAM
**Quality**control
******reads**with**N**errors
******reads**with**multiple**mappings
******strand**bias
******paired-end**insert
**Filtering:**by**number**of**errors,**number**of**hits
******Comparator:**stats,**intersection,**...
**Output:**BAM**file**plus**an**HTML**file**containing**statistics.
Identification**of**single**nucleotide**variants**and**indels**on**the**alignments**is**performed**using**the**Genome**Analysis**Toolkit**(GATK).**GATK**(2)**is**a**software**package**developed**at**the**Broad**Institute**to**analyze**high-throughput**sequencing**data.**The**toolkit**offers**a**wide**variety**of**tools,**with**a**primary**focus**on**variant**discovery**and**genotyping**as**well**as**strong**emphasis**on**data**quality**assurance.
VCF**(3)**is**a**standardized**format**for**storing**the**most**prevalent**types**of**sequence**variation,**including**SNPs,**indels**and**larger**structural**variants,**together**with**rich**annotations.**The**format**was**developed**with**the**primary**intention**to**represent**human**genetic**variation,**but**its**use**is**not**restricted**>to**diploid**genomes**and**can**be**used**in**different**contexts**as**well.**Its**flexibility**and**user**extensibility**allows**representation**of**a**wide**variety**of**genomic**variation**with**respect**to**a**single**reference**sequence.
A**VCF**file**consists**of**a**header**section**and**a**data**section.**The**header**contains**an**arbitrary**number**of**metainformation**lines,**each**starting**with**characters**‘##’,**and**a**TAB**delimited**field**definition**line,**starting**with**a**single**‘#’**character.**The**meta-information**header**lines**provide**a**standardized**description**of**tags**and**annotations**used**in**the**data**section.**The**use**of**meta-information**allows**the**information**stored**within**a**VCF**file**to**be**tailored**to**the**dataset**in**question.**It**can**be**also**used**to**provide**information**about**the**means**of**file**creation,**date**of**creation,**version**of**the**reference**sequence,**software**used**and**any**other**information**relevant**to**the**history**of**the**file.**The**field**definition**line**names**eight**mandatory**columns,**corresponding**to**data**columns**representing**the**chromosome**(CHROM),**a**1-based**position**of**the**start**of**the**variant**(POS),**unique**identifiers**of**the**variant**(ID),**the**reference**allele**(REF),**a**comma**separated**list**of**alternate**non-reference**alleles**(ALT),**a**phred-scaled**quality**score**(QUAL),**site**filtering**information**(FILTER)**and**a**semicolon**separated**list**of**additional,**user**extensible**annotation**(INFO).**In**addition,**if**samples**are**present**in**the**file,**the**mandatory**header**columns**are**followed**by**a**FORMAT**column**and**an**arbitrary**number**of**sample**IDs**that**define**the**samples**included**in**the**VCF**file.**The**FORMAT**column**is**used**to**define**the**information**contained**within**each**subsequent**genotype**column,**which**consists**of**a**colon**separated**list**of**fields.**For**example,**the**FORMAT**field**GT:GQ:DP**in**the**fourth**data**entry**of**Figure**1a**indicates**that**the**subsequent**entries**contain**information**regarding**the**genotype,**genotype**quality**and**read**depth**for**each**sample.**All**data**lines**are**TAB**delimited**and**the**number**of**fields**in**each**data**line**must**match**the**number**of**fields**in**the**header**line.**It**is**strongly**recommended**that**all**annotation**tags**used**are**declared**in**the**VCF**header**section.
**Figure**4****.**(a)**Example**of**valid**VCF.**The**header**lines**##fileformat**and**#CHROM**are**mandatory,**the**rest**is**optional**but**strongly**recommended.**Each**line**of**the**body**describes**variants**present**in**the**sampled**population**at**one**genomic**position**or**region.**All**alternate**alleles**are**listed**in**the**ALT**column**and**referenced**from**the**genotype**fields**as**1-based**indexes**to**this**list;**the**reference**haplotype**is**designated**as**0.**For**multiploid**data,**the**separator**indicates**whether**the**data**are**phased**(|)**or**unphased**(/).**Thus,**the**two**alleles**C**and**G**at**the**positions**2**and**5**in**this**figure**occur**on**the**same**chromosome**in**SAMPLE1.**The**first**data**line**shows**an**example**of**a**deletion**(present**in**SAMPLE1)**and**a**replacement**of**two**bases**by**another**base**(SAMPLE2);**the**second**line**shows**a**SNP**and**an**insertion;**the**third**a**SNP;**the**fourth**a**large**structural**variant**described**by**the**annotation**in**the**INFO**column,**the**coordinate**is**that**of**the**base**before**the**variant.**(b–f**)**Alignments**and**VCF**representations**of**different**sequence**variants:**SNP,**insertion,**deletion,**replacement,**and**a**large**deletion.**The**REF**columns**shows**the**reference**bases**replaced**by**the**haplotype**in**the**ALT**column.**The**coordinate**refers**to**the**first**reference**base.**(g)**Users**are**advised**to**use**simplest**representation**possible**and**lowest**coordinate**in**cases**where**the**position**is**ambiguous.
The**functional**consequences**of**every**variant**found**are**then**annotated**using**the**HPG-Variant**software,**which**extracts**from**CellBase,**the**Knowledge**database,**all**the**information**relevant**on**the**predicted**pathologic**effect**of**the**variants.
VARIANT**(VARIant**Analysis**Tool)**(4)**reports**information**on**the**variants**found**that**include**consequence**type**and**annotations**taken**from**different**databases**and**repositories**(SNPs**and**variants**from**dbSNP**and**1000**genomes,**and**disease-related**variants**from**the**Genome-Wide**Association**Study**(GWAS)**catalog,**Online**Mendelian**Inheritance**in**Man**(OMIM),**Catalog**of**Somatic**Mutations**in**Cancer**(COSMIC)**mutations,**etc.**VARIANT**also**produces**a**rich**variety**of**annotations**that**include**information**on**the**regulatory**(transcription**factor**or**miRNAbinding**sites,**etc.)**or**structural**roles,**or**on**the**selective**pressures**on**the**sites**affected**by**the**variation.**This**information**allows**extending**the**conventional**reports**beyond**the**coding**regions**and**expands**the**knowledge**on**the**contribution**of**non-coding**or**synonymous**variants**to**the**phenotype**studied.
**Output:****The**output**of**this**step**is**the**Variant**Calling**Format**(VCF)**file,**which**contains**changes**with**respect**to**the**reference**genome**with**the**corresponding**QC**and**functional**annotations.
CellBase(5)**is**a**relational**database**integrates**biological**information**from**different**sources**and**includes:
We**took**genome**sequences,**genes,**transcripts,**exons,**cytobands**or**cross**references**(xrefs)**identifiers**(IDs)**from**Ensembl**(6).**Protein**information**including**sequences,**xrefs**or**protein**features**(natural**variants,**mutagenesis**sites,**post-translational**modifications,**etc.)**were**imported**from**UniProt**(7).
CellBase**imports**miRNA**from**miRBase**(8);**curated**and**non-curated**miRNA**targets**from**miRecords**(9),**miRTarBase**(10),
TargetScan(11)**and**microRNA.org**(12)**and**CpG**islands**and**conserved**regions**from**the**UCSC**database**(13).
OBO**Foundry**(14)**develops**many**biomedical**ontologies**that**are**implemented**in**OBO**format.**We**designed**a**SQL**schema**to**store**these**OBO**ontologies**and**30**ontologies**were**imported.**OBO**ontology**term**annotations**were**taken**from**Ensembl**(6).**InterPro**(15)**annotations**were**also**imported.
CellBase**includes**SNPs**from**dbSNP**(16)^;**SNP**population**frequencies**from**HapMap**(17),**1000**genomes**project**(18)**and**Ensembl**(6);**phenotypically**annotated**SNPs**were**imported**from**NHRI**GWAS**Catalog**(19),HGMD**(20),**Open**Access**GWAS**Database**(21),**UniProt**(7)**and**OMIM**(22);**mutations**from**COSMIC**(23)**and**structural**variations**from**Ensembl**(6).
We**also**import**systems**biology**information**like**interactome**information**from**IntAct**(24).**Reactome**(25)**stores**pathway**and**interaction**information**in**BioPAX**(26)**format.**BioPAX**data**exchange**format**enables**the**integration**of**diverse**pathway
resources.**We**successfully**solved**the**problem**of**storing**data**released**in**BioPAX**format**into**a**SQL**relational**schema,**which**allowed**us**importing**Reactome**in**CellBase.
###**[Diagnostic**Component**(TEAM)](diagnostic-component-team/)
###**[Priorization**Component**(BiERApp)](priorization-component-bierapp/)
This**command**will**load**python/2.7.5**module**and**all**the**required**modules**(hpg-aligner,**gatk,**etc)
If**we**launch**ngsPipeline**with**‘-h’,**we**will**get**the**usage**help:
********$**ngsPipeline**-h
********Usage:**ngsPipeline.py**[-h]**-i**INPUT**-o**OUTPUT**-p**PED**--project**PROJECT**--queue
************QUEUE**[--stages-path**STAGES_PATH]**[--email**EMAIL]
**********[--prefix**PREFIX]**[-s**START]**[-e**END]**--log
********Python**pipeline
********optional**arguments:
************-h,**--help**show**this**help**message**and**exit
************-i**INPUT,**--input**INPUT
************-o**OUTPUT,**--output**OUTPUT
****************Output**Data**directory
************-p**PED,**--ped**PED**********Ped**file**with**all**individuals
************--project**PROJECT**********Project**Id
************--queue**QUEUE******************Queue**Id
************--stages-path**STAGES_PATH
****************Custom**Stages**path
************--email**EMAIL******************Email
************--prefix**PREFIX**************Prefix**name**for**Queue**Jobs**name
************-s**START,**--start**START
****************Initial**stage
************-e**END,**--end**END**********Final**stage
************--log**************Log**to**file
*************-i,**--input.**The**input**data**directory.**This**directory**must**to**have**a**special**structure.**We**have**to**create**one**folder**per**sample**(with**the**same**name).**These**folders**will**host**the**fastq**files.**These**fastq**files**must**have**the**following**pattern**“sampleName”**+**“_”**+**“1**or**2”**+**“.fq”.**1**for**the**first**pair**(in**paired-end**sequences),**and**2**for**the
second**one.
*************-o**,**--output.**The**output**folder.**This**folder**will**contain**all**the**intermediate**and**final**folders.**When**the**pipeline**will**be**executed**completely,**we**could**remove**the**intermediate**folders**and**keep**only**the**final**one**(with**the**VCF**file**containing**all**the**variants)
*************-p**,**--ped*.**The**ped**file**with**the**pedigree.**This**file**contains**all**the**sample**names.**These**names**must**coincide**with**the**names**of**the**input**folders.**If**our**input**folder**contains**more**samples**than**the**.ped**file,**the**pipeline**will**use**only**the**samples**from**the**.ped**file.
*************-s,**--start**&**-e,**--end.****Initial**and**final**stage.**If**we**want**to**launch**the**pipeline**in**a**specific**stage**we**must**use**-s.**If**we**want**to**end**the**pipeline**in**a**specific**stage**we**must**use**-e.
*************--log*.**Using**log**argument**NGSpipeline**will**prompt**all**the**logs**to**this**file.
*************--project*>.**Project**ID**of**your**supercomputer**allocation.
*************--queue*.**[Queue](../../resource-allocation-and-job-execution/introduction.html)**to**run**the**jobs**in.
Input,**output**and**ped**arguments**are**mandatory.**If**the**output**folder**does**not**exist,**the**pipeline**will**create**it.
/apps/bio/omics/1.0/sample_data/**>:
********/apps/bio/omics/1.0/sample_data
********└──**data
****************├──**file.ped
****************├──**sample1
****************│******├──**sample1_1.fq
****************│******└──**sample1_2.fq
****************└──**sample2
****├──**sample2_1.fq
****└──**sample2_2.fq
********#family_ID**sample_ID**parental_ID**maternal_ID**sex**phenotype
********FAM**sample_A**0**0**1**1
********FAM**sample_B**0**0**2**2
Now,**lets**load**the**NGSPipeline**module**and**copy**the**sample**data**to**a**[scratch**directory](../../storage/storage/):
********$**module**load**ngsPipeline
********$**mkdir**-p**/scratch/$USER/omics/results
********$**cp**-r**/apps/bio/omics/1.0/sample_data**/scratch/$USER/omics/
Now,**we**can**launch**the**pipeline**(replace**OPEN-0-0**with**your**Project**ID):
********$**ngsPipeline**-i**/scratch/$USER/omics/sample_data/data**-o**/scratch/$USER/omics/results**-p**/scratch/$USER/omics/sample_data/data/file.ped**--project**OPEN-0-0**--queue**qprod
This**command**submits**the**processing**[jobs**to**the**queue](../../resource-allocation-and-job-execution/job-submission-and-execution.html).
If**we**want**to**re-launch**the**pipeline**from**stage**4**until**stage**20**we**should**use**the**next**command:
********$**ngsPipeline**-i**/scratch/$USER/omics/sample_data/data**-o**/scratch/$USER/omics/results**-p**/scratch/$USER/omics/sample_data/data/file.ped**-s**4**-e**20**--project**OPEN-0-0**--queue**qprod
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##**Details**on**the**Pipeline
The**pipeline**calls**the**following**tools
**[fastqc](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/),**quality**control**tool**for**high**throughput**sequence**data.
**[gatk](https://www.broadinstitute.org/gatk/),**The**Genome**Analysis**Toolkit**or**GATK**is**a**software**package**developed**at
************the**Broad**Institute**to**analyze**high-throughput**sequencing**data.**The**toolkit**offers**a**wide**variety**of**tools,**with**a**primary**focus**on**variant**discovery**and**genotyping**as**well**as**strong**emphasis**on**data**quality**assurance.**Its**robust**architecture,**powerful**processing**engine**and**high-performance**computing**features**make**it**capable**of**taking**on**projects**of**any**size.
**[hpg-aligner](https://github.com/opencb-hpg/hpg-aligner),**HPG**Aligner**has**been**designed**to**align**short**and**long**reads**with**high**sensitivity,**therefore**any**number**of**mismatches**or**indels**are**allowed.**HPG**Aligner**implements**and**combines**two**well**known**algorithms:**_Burrows-Wheeler**Transform_**(BWT)**to**speed-up**mapping**high-quality**reads,**and**_Smith-Waterman_>**(SW)**to**increase**sensitivity**when**reads**cannot**be**mapped**using**BWT.
**[hpg-fastq](http://docs.bioinfo.cipf.es/projects/fastqhpc/wiki),**a**quality**control**tool**for**high**throughput**sequence**data.
**[hpg-variant](http://docs.bioinfo.cipf.es/projects/hpg-variant/wiki),**The**HPG**Variant**suite**is**an**ambitious**project**aimed**to**provide**a**complete**suite**of**tools**to**work**with**genomic**variation**data,**from**VCF**tools**to**variant**profiling**or**genomic**statistics.**It**is**being**implemented**using**High**Performance**Computing**technologies**to**provide**the**best**performance**possible.
**[picard](http://picard.sourceforge.net/),**Picard**comprises**Java-based**command-line**utilities**that**manipulate**SAM**files,**and**a**Java**API**(HTSJDK)**for**creating**new**programs**that**read**and**write**SAM**files.**Both**SAM**text**format**and**SAM**binary**(BAM)**format**are**supported.
**[samtools](http://samtools.sourceforge.net/samtools-c.shtml),**SAM**Tools**provide**various**utilities**for**manipulating**alignments**in**the**SAM**format,**including**sorting,**merging,**indexing**and**generating**alignments**in**a**per-position**format.
**[snpEff](http://snpeff.sourceforge.net/),**Genetic**variant**annotation**and**effect**prediction**toolbox.
This**listing**show**which**tools**are**used**in**each**step**of**the**pipeline
**stage-00:**fastqc
**stage-01:**hpg_fastq
**stage-02:**fastqc
**stage-03:**hpg_aligner**and**samtools
**stage-04:**samtools
**stage-05:**samtools
**stage-06:**fastqc
**stage-07:**picard
**stage-08:**fastqc
**stage-09:**picard
**stage-10:**gatk
**stage-11:**gatk
**stage-12:**gatk
**stage-13:**gatk
**stage-14:**gatk
**stage-15:**gatk
**stage-16:**samtools
**stage-17:**samtools
**stage-18:**fastqc
**stage-19:**gatk
**stage-20:**gatk
**stage-21:**gatk
**stage-22:**gatk
**stage-23:**gatk
**stage-24:**hpg-variant
**stage-25:**hpg-variant
**stage-26:**snpEff
**stage-27:**snpEff
**stage-28:**hpg-variant
##**Interpretation
The**output**folder**contains**all**the**subfolders**with**the**intermediate**data.**This**folder**contains**the**final**VCF**with**all**the**variants.**This**file**can**be**uploaded**into**[TEAM](diagnostic-component-team.html)**by**using**the**VCF**file**button.**It**is**important**to**note**here**that**the**entire**management**of**the**VCF**file**is**local:**no**patient’s**sequence**data**is**sent**over**the**Internet**thus**avoiding**any**problem**of**data**privacy**or**confidentiality.
![TEAM**upload**panel.**Once**the**file**has**been**uploaded,**a**panel**must**be**chosen**from**the**Panel**list.**Then,**pressing**the**Run**button**the**diagnostic**process**starts.]\((../../../img/fig7.png)
**Figure**7.****_TEAM**upload**panel._**_Once**the**file**has**been**uploaded,**a**panel**must**be**chosen**from**the**Panel_**list.**Then,**pressing**the**Run**button**the**diagnostic**process**starts.
Once**the**file**has**been**uploaded,**a**panel**must**be**chosen**from**the**Panel**list.**Then,**pressing**the**Run**button**the**diagnostic**process**starts.**TEAM**searches**first**for**known**diagnostic**mutation(s)**taken**from**four**databases:**HGMD-public**(20),**[HUMSAVAR](http://www.uniprot.org/docs/humsavar),**ClinVar**(29)**and**COSMIC**(23).
**Figure**7.****The**panel**manager.**The**elements**used**to**define**a**panel**are**(**A****)**disease**terms,**(**B****)**diagnostic**mutations**and**(**C****)**genes.**Arrows**represent**actions**that**can**be**taken**in**the**panel**manager.**Panels**can**be**defined**by**using**the**known**mutations**and**genes**of**a**particular**disease.**This**can**be**done**by**dragging**them**to**the****Primary**Diagnostic****box**(action****D****).**This**action,**in**addition**to**defining**the**diseases**in**the****Primary**Diagnostic****box,**automatically**adds**the**corresponding**genes**to**the****Genes****box.**The**panels**can**be**customized**by**adding**new**genes**(action****F****)**or**removing**undesired**genes**(action****G**).**New**disease**mutations**can**be**added**independently**or**associated**to**an**already**existing**disease**term**(action****E****).**Disease**terms**can**be**removed**by**simply**dragging**them**back**(action****H****).
For**variant**discovering/filtering**we**should**upload**the**VCF**file**into**BierApp**by**using**the**following**form:
\*\*
**Figure**8****.**\*BierApp**VCF**upload**panel.**It**is**recommended**to**choose**a**name**for**the**job**as**well**as**a**description**\*\*.
Each**prioritization**(‘job’)**has**three**associated**screens**that**facilitate**the**filtering**steps.**The**first**one,**the**‘Summary’**tab,**displays**a**statistic**of**the**data**set**analyzed,**containing**the**samples**analyzed,**the**number**and**types**of**variants**found**and**its**distribution**according**to**consequence**types.**The**second**screen,**in**the**‘Variants**and**effect’**tab,**is**the**actual**filtering**tool,**and**the**third**one,**the**‘Genome**view’**tab,**offers**a**representation**of**the**selected**variants**within**the**genomic**context**provided**by**an**embedded**version**of**the**Genome**Maps**Tool**(30).
**Figure**9****.**This**picture**shows**all**the**information**associated**to**the**variants.**If**a**variant**has**an**associated**phenotype**we**could**see**it**in**the**last**column.**In**this**case,**the**variant**7:132481242**CT**is**associated**to**the**phenotype:**large**intestine**tumor.
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