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GBS Bioinformatics Pipeline(s) Overview
Getting from sequence files to genotypes.
Pipeline Coding: Ed Buckler Jeff Glaubitz James Harriman
Presentation: Rob Elshire With supporting information from the coders.
Three Pipelines
• Discovery Pipeline – Requires a reference genome
– Multiple steps to get to genotypes
– Hands on tutorial is based on this pipeline
• Production Pipeline – Uses information from Discovery Pipeline
– One step from sequence to genotypes
• UNEAK Pipeline – For species without a reference genome
– Fei Lu will present this tomorrow at 9:30
Vocabulary • Sequence File
– Text file containing DNA sequence and supplemental information from the Illumina Platform.
• Key File – Text file used to assign a GBS Bar Code to a Taxa
• GBS Tag – DNA sequence consisting of a cut site remnant and
additional sequence.
• GBS Bar Code – A short known sequence of DNA used to assign a GBS
Tag to its original Taxa
• Taxa – An individual sample
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
HWI-ST397 0 3 68 15896 200039 0 1 GTCGATTCTGCTGACTTCATGGCTTCTGTTGACGACGATGTGGAACGAGCTGTTGTTGAAACTGATGAGGTTGCTGAGATCGGAAGAGCGGTTCAGCAGG HWI-ST397 0 3 68 15960 200043 0 1 GAGAATCAGCTTTTCCAACACCTTGAGTTTGAGTATGCGATGACAGTTACTCTTACTGTCCATTGTCAGCATTGCCAGAGCTTGACCAGCTGAGATCGGA HWI-ST397 0 3 68 15831 200053 0 1 ATGTACTGCACCGTTGCAAGCGAGCACCACCAAGCGGCGGTATGCACTTTGCAATATGTAGCTAGAATAGGATTTTCAGGTGATTAGGAGCGTAAAAAAG HWI-ST397 0 3 68 15867 200049 0 1 CCAGCTCAGCCTGCATTCTTTCAAAAACTTCCAATGCCTCTCTTGGCCTAGCATTTTGGGCATACCCTGTGACCATTGCTGTCCATGCCACCATATCCTT HWI-ST397 0 3 68 15943 200048 0 1 GATTTTACTGCACATCGGTCTTGTCACACCAGCTATACCTGTAGAGTTGCCTTCCACAGTTGTAGAGATCGGAAGAGCGGTTCAGCGGGACTGCCGAGAA HWI-ST397 0 3 68 15812 200062 0 1 TCACCCAGCATCACGCCCCTTCACATCCAGTAAAACCCCTGAATGATGTGCTGTCACTGTTTGATATACAGTTGTTAACGTGAGGACGGGCTTTGAAGGA HWI-ST397 0 3 68 15888 200067 0 1 CTTGACTGCCACCATGAATATGTGTTCCAAGTGCCACAAGGACTTGGCCCTGAAGCAAGAACAAGCCAAACTTGCAGAGATCGGAAGAGCGGTTCAGCAG HWI-ST397 0 3 68 15969 200067 0 1 CCACAACTGCTCCATCTTTTCCATGAGACATTGCTCCCGCCATTGCACCCTTGGCATCAGCAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG HWI-ST397 0 3 68 15786 200078 0 1 GTATTCTGCACACGAATCAGCTGAGACACCAATTGGGCATGAATCAAATGGCGCCATTGCCGGGGATCGAACCCCGAATCAAATGGTGCCATTGCCACTG HWI-ST397 0 3 68 15830 200072 0 1 AATATGCCAGCAGTTAAGAGAGTTCAAGATCCAGGGCTCATATTCAGTCACCTATATCAATTTCGAAATGGATTTCCAGGGTTTTAAGAGCCTAACAAAG HWI-ST397 0 3 68 15863 200073 0 1 CTCCCTGCGGGTGCGCGCGACCCATCTTCAGTTGGAGCGTCTATCGGCGTTGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTA HWI-ST397 0 3 68 15762 200088 0 1 TGGTACGTCTGCGGAATGGCGTTTTTTATGCCTTAGTGGTTCGCAGAGCATTTGGCAGCTGAGATGGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGAT HWI-ST397 0 3 68 15903 200085 0 1 GGACCTACTGCCCAAGAACGGCTCACCCATCATCCGCTTTCTTCACCTTCCGTCTTCTTTGGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGAC HWI-ST397 0 3 68 15921 200082 0 1 GAGAATCAGCGTGTACGGGGCACGGGGTGACTGCTGTTGCGTGCGAGGGCTGAGATCGGAAGAGCGGTTCAGCAGGAGTGCCGAGACCGATCTCGTATGC HWI-ST397 0 3 68 15984 200085 0 1 TTCTCCAGCCGCATGGGCCGGAGACCAGAGAGGCCTCCCCAGGATTTGCACGATAGACCACGACTTATGGACGATTGGGAAGCCCTTGTTGGAAGGAAAT HWI-ST397 0 3 68 15788 200096 0 1 GCGTCAGCAAATGCCCCAACAGCCAAGTCAGCAATTGCCTCAGCAACTTGGGCCACAAACACCACAGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCC HWI-ST397 0 3 68 15842 200099 0 1 TAGGCCATCAGCTGACTTCCCGGGTGTGGAGAAAAGAGGGCCCCTCACTTCTCTCAAGTGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCGGAGACCG HWI-ST397 0 3 68 15876 200105 0 1 GGACCTACTGCCGGCGGGACGAAAGCGGTTGTTGAATGATGGGGGTCACTAGGCCTTCCAGGGCCTTTAAGCGCGCGCTGAGATCGGAAGAGGGGTTCAG HWI-ST397 0 3 68 15937 200097 0 1 CTCCCTGTTGAAGCATGTGCAAAAGAGCTTGTTCTCGGCCTTCTTCAAGCCATTCTCTTGGCAGACGGCTTTGCCTAGAAGTTTCGCCCCATCACCCTTG HWI-ST397 0 3 68 15958 200102 0 1 CGCCTTATCTGCCCTCGCCGGTCATGGGGAGTGGTGCCCCTACCTCGGACAAGACAGATGCAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
Raw Sequence (Qseq)
HWI-ST397 0 3 68 15896 200039 0 1 GTCGATTCTGCTGACTTCATGGCTTCTGTTGACGACGATGTGGAACGAGCTGTTGTTGAAACTGATGAGGTTGCTGAGATCGGAAGAGCGGTTCAGCAGG HWI-ST397 0 3 68 15960 200043 0 1 GAGAATCAGCTTTTCCAACACCTTGAGTTTGAGTATGCGATGACAGTTACTCTTACTGTCCATTGTCAGCATTGCCAGAGCTTGACCAGCTGAGATCGGA HWI-ST397 0 3 68 15831 200053 0 1 ATGTACTGCACCGTTGCAAGCGAGCACCACCAAGCGGCGGTATGCACTTTGCAATATGTAGCTAGAATAGGATTTTCAGGTGATTAGGAGCGTAAAAAAG HWI-ST397 0 3 68 15867 200049 0 1 CCAGCTCAGCCTGCATTCTTTCAAAAACTTCCAATGCCTCTCTTGGCCTAGCATTTTGGGCATACCCTGTGACCATTGCTGTCCATGCCACCATATCCTT HWI-ST397 0 3 68 15943 200048 0 1 GATTTTACTGCACATCGGTCTTGTCACACCAGCTATACCTGTAGAGTTGCCTTCCACAGTTGTAGAGATCGGAAGAGCGGTTCAGCGGGACTGCCGAGAA HWI-ST397 0 3 68 15812 200062 0 1 TCACCCAGCATCACGCCCCTTCACATCCAGTAAAACCCCTGAATGATGTGCTGTCACTGTTTGATATACAGTTGTTAACGTGAGGACGGGCTTTGAAGGA HWI-ST397 0 3 68 15888 200067 0 1 CTTGACTGCCACCATGAATATGTGTTCCAAGTGCCACAAGGACTTGGCCCTGAAGCAAGAACAAGCCAAACTTGCAGAGATCGGAAGAGCGGTTCAGCAG HWI-ST397 0 3 68 15969 200067 0 1 CCACAACTGCTCCATCTTTTCCATGAGACATTGCTCCCGCCATTGCACCCTTGGCATCAGCAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG HWI-ST397 0 3 68 15786 200078 0 1 GTATTCTGCACACGAATCAGCTGAGACACCAATTGGGCATGAATCAAATGGCGCCATTGCCGGGGATCGAACCCCGAATCAAATGGTGCCATTGCCACTG HWI-ST397 0 3 68 15830 200072 0 1 AATATGCCAGCAGTTAAGAGAGTTCAAGATCCAGGGCTCATATTCAGTCACCTATATCAATTTCGAAATGGATTTCCAGGGTTTTAAGAGCCTAACAAAG HWI-ST397 0 3 68 15863 200073 0 1 CTCCCTGCGGGTGCGCGCGACCCATCTTCAGTTGGAGCGTCTATCGGCGTTGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTA HWI-ST397 0 3 68 15762 200088 0 1 TGGTACGTCTGCGGAATGGCGTTTTTTATGCCTTAGTGGTTCGCAGAGCATTTGGCAGCTGAGATGGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGAT HWI-ST397 0 3 68 15903 200085 0 1 GGACCTACTGCCCAAGAACGGCTCACCCATCATCCGCTTTCTTCACCTTCCGTCTTCTTTGGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGAC HWI-ST397 0 3 68 15921 200082 0 1 GAGAATCAGCGTGTACGGGGCACGGGGTGACTGCTGTTGCGTGCGAGGGCTGAGATCGGAAGAGCGGTTCAGCAGGAGTGCCGAGACCGATCTCGTATGC HWI-ST397 0 3 68 15984 200085 0 1 TTCTCCAGCCGCATGGGCCGGAGACCAGAGAGGCCTCCCCAGGATTTGCACGATAGACCACGACTTATGGACGATTGGGAAGCCCTTGTTGGAAGGAAAT HWI-ST397 0 3 68 15788 200096 0 1 GCGTCAGCAAATGCCCCAACAGCCAAGTCAGCAATTGCCTCAGCAACTTGGGCCACAAACACCACAGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCC HWI-ST397 0 3 68 15842 200099 0 1 TAGGCCATCAGCTGACTTCCCGGGTGTGGAGAAAAGAGGGCCCCTCACTTCTCTCAAGTGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCGGAGACCG HWI-ST397 0 3 68 15876 200105 0 1 GGACCTACTGCCGGCGGGACGAAAGCGGTTGTTGAATGATGGGGGTCACTAGGCCTTCCAGGGCCTTTAAGCGCGCGCTGAGATCGGAAGAGGGGTTCAG HWI-ST397 0 3 68 15937 200097 0 1 CTCCCTGTTGAAGCATGTGCAAAAGAGCTTGTTCTCGGCCTTCTTCAAGCCATTCTCTTGGCAGACGGCTTTGCCTAGAAGTTTCGCCCCATCACCCTTG HWI-ST397 0 3 68 15958 200102 0 1 CGCCTTATCTGCCCTCGCCGGTCATGGGGAGTGGTGCCCCTACCTCGGACAAGACAGATGCAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
Raw Sequence (Qseq)
Key File
Flowcell Lane Barcode DNASample LibraryPlate Row Column LibraryPrepID PlateName
81PVTABXX 2 CTCC Sample_1 1 A 1 1 Plate_A
81PVTABXX 2 TGCA Sample_2 1 A 2 2 Plate_A
81PVTABXX 2 ACTA Sample_3 1 A 3 3 Plate_A
81PVTABXX 2 CAGA Sample_4 1 A 4 4 Plate_A
81PVTABXX 2 AACT Sample_5 1 A 5 5 Plate_A
81PVTABXX 2 GCGT Sample_6 1 A 6 6 Plate_A
81PVTABXX 2 TGCGA Sample_7 1 A 7 7 Plate_A
81PVTABXX 2 CGAT Sample_8 1 A 8 8 Plate_A
81PVTABXX 2 CGCTT Sample_9 1 A 9 9 Plate_A
81PVTABXX 2 TCACC Sample_10 1 A 10 10 Plate_A
81PVTABXX 2 CTAGC Sample_11 1 A 11 11 Plate_A
81PVTABXX 2 ACAAA Sample_12 1 A 12 12 Plate_A
81PVTABXX 2 TTCTC Sample_13 1 B 1 13 Plate_A
81PVTABXX 2 AGCCC Sample_14 1 B 2 14 Plate_A
81PVTABXX 2 GTATT Sample_15 1 B 3 15 Plate_A
81PVTABXX 2 CTGTA Sample_16 1 B 4 16 Plate_A
81PVTABXX 2 ACCGT Sample_17 1 B 5 17 Plate_A
81PVTABXX 2 GTAA Sample_18 1 B 6 18 Plate_A
81PVTABXX 2 GGTTGT Sample_19 1 B 7 19 Plate_A
Chimeric (?) sequence
Rejected reads
‘Good’ read
Insert Barcode
Cut site
Short sequence
Adapter dimer
Insert Barcode adapter Cut site Common adapter Cut site
Insert Cut site 2nd Insert Barcode
Cut site
GBS Tags
Barcode
Cut site Common adapter
Trimmed reads
Insert Cut site
Insert Barcode
No Cut site
No Barcode
Insert Barcode
Cut site Common adapter Cut site
Chimeric (?) sequence
Rejected reads
‘Good’ read
Insert Barcode
Cut site
Short sequence
Adapter dimer
Insert Barcode adapter Cut site Common adapter Cut site
Insert Cut site 2nd Insert Barcode
Cut site
GBS Tags
Barcode
Cut site Common adapter
Trimmed reads
Insert Cut site
Insert Barcode
No Cut site
No Barcode
Insert Barcode
Cut site Common adapter Cut site
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
Tag Counts
• With information from the key file, each sequence file is processed, tags are identified and counted.
• If a tag is shorter than 64 bases it is padded.
• The tags and counts are put into a tag count file for each sequence file.
QseqToTagCountsPlugin / FastqToTagCountsPlugin
Master Tag Counts
• The individual tag count files are merged into a master tag count file.
• A minimum count is specified at the merge stage to exclude tags with low counts (likely sequencing errors).
MergeMultipleTagCountsPlugin
Conversion of Tags to Fastq
• Sequence aligners do not work with the tag count file format.
• In preparation for the alignment step, the tag count file is converted to fastq format.
TagCountsToFastqPlugin
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
Tag Alignment / TOPM
• The GBS pipeline uses an external aligner to do the initial alignment.
• The current version uses bowtie2 which produces the alignment in the SAM format.
• We convert the SAM file into our tags on physical map format (TOPM)
bowtie2
SAMConverterPlugin
TOPM
So Far We Have
• Identified and counted GBS tags.
• Converted tag counts file to fastq.
• Aligned the tags to a reference.
• Converted the alignment to TOPM.
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
Tags by Taxa
• In this step we identify which tags are present in which taxa.
– Original Sequence Files
– Key File
– Master Tag Count File
• Recently migrated to HDF5 file format.
– Efficient storage
– Large data sets
SeqToTBTHDF5Plugin
Tags By Taxa Additional Operations
• If many TBTs have been created they are merged into 1 TBT.
• Taxa that were sequenced multiple times are merged.
• The TBT table is pivoted in preparation for SNP calling.
ModifyTBTHDF5Plugin
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Sequence
TOPM
GBS Discovery Pipeline
SNP Calling
• Files used in SNP Calling – TOPM
– TBT
– Pedigree File (optional)
• Some Key Settings – mnF MinimumF (inbreeding coefficient)
– mnMAF Minimum Minor Allele Frequency
– mnMAC Minimum Minor Allele Count
– mnLCov Minimum Locus Coverage
TagsToSNPByAlignmentPlugin
HapMap rs# alleles chrom pos strand SgSBRIL067:633Y5AAXX:2:C9 SgSBRIL019:633Y5AAXX:2:C3 S1_2100 A/G 1 2100 + N N N N N N N R N A N S1_2163 T/C 1 2163 + N N N N N N T C T T N S1_13837 T/G 1 13837 + N N N N N N N G N N T S1_14606 C/T 1 14606 + N N C N N N T T T T C S1_2061 T/A 1 20601 + T N N N N N N A N N N S1_68332 C/T 1 68332 + N N N N N N N N N N N S1_68596 A/T 1 68596 + A N N N N N N N N A N S1_69309 G/A 1 69309 + N G N N N N N A N N N S1_79955 T/G 1 79955 + N T G T T N T T N N N S1_79961 T/G 1 79961 + N T T T T N T T N N N S1_80584 G 1 80584 + N N N N N N N N N N G S1_80647 C/T 1 80647 + N N N N N N N C N N C S1_81274 T/G 1 81274 + N N N N N N T G N N N S1_108834 G/A 1 108834 + N N N N N N N N N N N S1_112345 T/G 1 112345 + N N N N N N K T N N N S1_115359 C/T 1 115359 + N N N N N N T C N T S1_115362 T/C 1 115362 + N N N N N N N C N N N S1_115405 G/A 1 115405 + G G A N N G G G G N S1_115516 T/G 1 115516 + N N T N N N T T N N T S1_116694 A/G 1 116694 + N A G N N N G A N N N S1_119016 C/T 1 119016 + N N N N C N N C N N N S1_155366 T/C 1 155366 + N T N N N N
Production Pipeline
Why another pipeline?
• The last maize build (30000 taxa) with the discovery pipeline took over 3 months.
• Most common alleles have been identified after the first few discovery builds.
• Use the information from the discovery pipeline to call SNPs in new runs quickly.
• Improve efficiency and automate.
GBS Discovery pipeline
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
TOPM
GBS Discovery pipeline
Discovery
Tag Counts
SNP Caller
Tags by Taxa
Fastq
TOPM
Genotypes
Filtered Genotypes
GBS Bioinformatics Pipelines
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
TOPM
Fastq
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
TOPM
Fastq
TagsOnPhysicalMap (TOPM)
GBS Bioinformatics Pipelines
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
Filtered Genotypes
TOPM
Fastq
GBS Bioinformatics Pipelines
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
Fastq
Filtered Genotypes
TOPM TOPM
GBS Bioinformatics Pipelines
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
Fastq
Filtered Genotypes
TOPM TOPM
GBS Bioinformatics Pipelines
Discovery
Tag Counts
SNP Caller
Genotypes
Tags by Taxa
Fastq
Production
Fastq
Filtered Genotypes
TOPM TOPM
Genotypes
Running the Production Pipeline
• Required Files: – Sequence file (fastq or qseq)
– Key file
– Production TOPM
• TASSEL 3 Standalone & RawReadsToHapMapPlugin
• Running the Pipeline: – One lane processed at a time
– HapMap files by chromosome
• ~7 minutes
Testing Production Pipeline
• Compared HapMap files produced by Discovery Pipeline and Production Pipeline
• Site Comparison:
– Discovery 48,139
– Production 47,676
– Difference due to maximum 8 alleles
• 99.98% correlation of genetic distance matrices
Next Steps In Pipeline Development
• Hierarchical Data Format – supports very large data sets and complex data structures.
• Working to fuse TOPM, TBT, Keyfile, and Pedigree File into one HDF5 repository.
• Continued improvements to SNP caller.
• Ability to use tags not present in the reference.
