HANDS ON EXERCISE – GBS WORKSHOP

ROB ELSHIRE, JEFF GLAUBITZ AND KATIE HYMA

WARNING: SMART CHOICES V. DEFAULTS V. TUTORIAL SETTINGS

The default settings for the steps in the GBS pipeline as well as those in this tutorial are not appropriate for your real life data. This tutorial is intended to get you familiar with the mechanics of running the pipeline. It is not meant as a guide to running the pipeline on your unique set of data. To analyze your data, you will need to be familiar with the genetics of your system and samples as well as the settings for each of the steps. Tuning the pipeline is an iterative process and best done with a data set for which you can have testable expectations of the outcome (e.g. a mapping population). The pipeline will give you SNP markers at the end, but it is up to you to make sure that they are good markers.

UNIX SHELL COMMANDS

ls – list directory contentscd – change dirctoryless (filename) -- display contents of a text file

TASSEL PIPELINE AND XML FILES

XML files are structured text files. In this case they are used to store configuration information for running TASSEL plugins.

You can open up the xml file with less or a text editor.

You can regenerate the command line like this:

./run_pipeline.pl -translateXML config.xml

EY Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM

Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM

COUNT GBS TAGScd /mnt/workshop/data/02_TagCounts/01_IndividualTagCounts/

run_pipeline.pl -Xms512m -Xmx1g -configFile ./FastqToTagCounts.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><FastqToTagCountPlugin><i>../../01_RawSequence</i><o>./</o><k>../../50_KeyFiles/Pipeline_Testing_key.txt</k><e>ApeKI</e><s>3000000</s><c>1</c></FastqToTagCountPlugin></fork1><runfork1/>

</TasselPipeline>

MERGE TAG COUNT FILES

cd /mnt/workshop/data/02_TagCounts/02_MergedTagCounts

run_pipeline.pl -Xms512m -Xmx1g -configFile ./MergeTagCounts.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><MergeMultipleTagCountPlugin><i>../01_IndividualTagCounts</i><o>./GBS_Workshop_Maize.cnt</o><c>5</c></MergeMultipleTagCountPlugin></fork1><runfork1/>

</TasselPipeline>

Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM

CONVERT TO FASTQ

cd /mnt/workshop/data/02_TagCounts/03_TagCountToFastq

run_pipeline.pl -Xms512m -Xmx1g -configFile TagCountToFastq.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><TagCountToFastqPlugin><i>../02_MergedTagCounts/GBS_Workshop_Maize.cnt</i><o>./GBS_Workshop_Maize.fq.gz</o><c>5</c></TagCountToFastqPlugin></fork1><runfork1/>

</TasselPipeline>

ALIGN GBS TAGS TO REFERENCE GENOME

cd /mnt/workshop/data/03_SAM/

bowtie2 -M 4 -p 15 --very-sensitive-local -x ../53_AlignerIdices/GBS_Workshop_Maize -U ../02_TagCounts/03_TagCountToFastq/GBS_Workshop_Maize.fq-S GBS_Workshop_Maize.sam

CONVERT SAM TO TAGS ON PHYSICAL MAP (TOPM)

cd /mnt/workshop/data/04_TOPM/

run_pipeline.pl -Xms512m -Xmx1g -configFile SAMConverter.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><SAMConverterPlugin><i>../03_SAM/GBS_Workshop_Maize.sam</i><o>./GBS_Workshop_Maize.topm</o></SAMConverterPlugin></fork1><runfork1/>

</TasselPipeline>

Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM

MATCH TAGS TO SAMPLES (TAXA)cd /mnt/workshop/data/05_TBT/01_IndividualTBT

run_pipeline.pl -Xms512m -Xmx1g -configFile SeqToTBTHDF5Plugin.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><SeqToTBTHDF5Plugin><i>../../01_RawSequence</i><k>../../50_KeyFiles/Pipeline_Testing_key.txt</k><e>ApeKI</e><o>./GBS_Workshop_Maize.h5</o><s>100000000</s><L>GBS_Workshop_MaizeTBTHDF5_Log.txt</L><t>../../02_TagCounts/02_MergedTagCounts/GBS_Workshop_Maize.cnt</t></SeqToTBTHDF5Plugin></fork1><runfork1/>

</TasselPipeline>

PIVOT THE TBT TO PREPARE FOR SNP CALLING

cd /mnt/workshop/data/05_TBT/04_PivotMergedTaxaTBT

run_pipeline.pl -Xms512m -Xmx1g -configFile ./PivotTaxaTBTHDF5.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><ModifyTBTHDF5Plugin><o>../01_IndividualTBT/GBS_Workshop_Maize.h5</o><p>./PivotTaxaTBTHDF5.h5</p><c></c></ModifyTBTHDF5Plugin></fork1><runfork1/>

</TasselPipeline>

Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM

CALL SNPS cd /mnt/workshop/data/06_HapMap/

run_pipeline.pl -Xms512m -Xmx1g -configFile ./SNP_Caller.xml

<?xml version="1.0" encoding="UTF-8" standalone="no"?><TasselPipeline>

<fork1><TagsToSNPByAlignmentPlugin><i>../05_TBT/04_PivotMergedTaxaTBT/PivotTaxaTBTHDF5.h5</i><o>./GBS_Workshop_Maize_chr+.hmp.txt</o><m>../04_TOPM/GBS_Workshop_Maize.topm</m><mnF>0.8</mnF><mnMAF>0.02</mnMAF><mnMAC>100000</mnMAC><sC>9</sC><eC>10</eC></TagsToSNPByAlignmentPlugin></fork1><runfork1/>

</TasselPipeline>

Discovery

Tag Counts

SNP Caller

Genotypes

Tags by Taxa

Sequence

TOPM