ion torrent read mapping and variant caller
2
0
Entering edit mode
7.9 years ago
bNw • 0

Hello,

i have sample data in fastq format and need to find the variants using the ion torrent suite. i applied the below steps but got "TVC ERROR:read group 4 does not have a flow order." while running variant caller command line . also i tried to use the demo ecoli from the torrent server but get empty vcf files

  1. mapping using TMAP command line, mapall on stage map4
  2. sort and index the bam file

  3. run variant_caller_pipeline.py on the bam file and the reference

    /results/plugins/variantCaller/bin/variant_caller_pipeline.py --input-bam /home/ionadmin/Downloads/R_2011_04_07_12_44_38_user_CB1-42-r9723-314wfa-tl_ecoli_aligmnet.bam --reference-fasta e_coli_dh10b.fasta

    ERROR returned

    TargetsManager: No targets file specified, processing entire reference
SampleManager: Found 1 read group(s) and 1 sample(s).
SampleManager: Primary sample "20" (default) present in 1 read group(s)
**TVC ERROR: read group 4 does not have a flow order.**
 ERROR: command failed with status 1

from torrent server

i did reanalysis to the demo ecoli sample but it generate empty vcf file also the same results if i run the tvc on bam file from the server

part of the sample :

@D8GEU:00484:01919
CGGCAATGTTGCACCGTTTGCTGCATGATATTGAAAAAAATATCACCAAATAAAAAACGCCTTAGTAAGTATTTTTCAGCTTTTCATTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATTAAAAAAAAGAGTGTCTGATAGCAGCTTCTGAACTGGTTACCTGCCGTGAGTAAATTAAAATTTTATTGACTTAGGTCACTAAATACTTTAACCAATATAGGCATAGCGCACAGACAGATAAAAATTACAGAGTACACAAACATCCATGAAACGCATTAGCACCACCATTACCACCACCATCACCTTACCACAGGTAACGGTGCGGGCTGACGCGACAGGAACACAGAAAAAGCCCGCACCTGA
+D8GEU:00484:01919
=<6<D8<=D>CCEE@DDK<?>ECC?<<<<=0===AAAMM-00.=??:>?0::????*:5:2:2:::::??:::::-::CEMGC4CCD@C????:::?:??E8>>AEEEDDCCDEEFFBB;::4:::::::&=DFGGDGKGE???LEDDDACCCDADAA:A5:::@@E9>>>>DDED>E4:::/:::/::2::BD<::4:::555:6ABBCC0:4:2:4:::::2::::6666:;;>>>666:::::-6366:6::6:?66:616C660:66;;,6666606>>;;8666;;8==@DE9?;066666066>>6666606>??D066666,6666666.....).28..<A;===0...).8..)...
@D8GEU:00695:01775
CCACCAAATAAAAAACGCCTTAGTAAGTATTTTTCAGCTTTTCATTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATTAAAAAAAGAGTGTCTGATAGCAGCCTTCTGAACTGGTTACCCTGCCGTGAGTAAAATTAAAATTTTATTGACTTAGGTCACTAAAATACTTTAACCAATATAGGCATAGCGCACAGACAGATAAAAATTACAGAGTACACAACATCCATGAAACGCATTAGCACCACCATTACCACC
+D8GEU:00695:01775
505@0..)......(...).).8889BB:::::-66B666<)6>C46:6655...)...).@3:6666666>666660660666666)666666>88....88660606>650555060655,5550666666666)60666)666)666666>6>>??>>>6666)66666,606060.....)..8.566=88........8888(.).88.6<@>88..)....)5555=6566660666..)..)..)..2..)

Bam file header generated from the torrent server

@HD    VN:1.4    SO:coordinate
@SQ    SN:gi|170079663|ref|NC_010473.1|    LN:4686137
@RG    ID:SEZXA    CN:TorrentServer/PGM_test    DT:2016-06-18T04:50:19    FO:TACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACGTACGTCTGAGCATCGATCGATGTACAGCTACG    KS:TCAG    PG:tmap    PL:IONTORRENT    PU:pgm/314R    SM:e5272-wfa-l165
@PG    ID:bc    PN:BaseCaller    CL:BaseCaller --barcode-filter 0.01 --barcode-filter-minreads 20 --phred-table-file /opt/ion/config/phredTable.314.B5.h5 --phase-estimation-file basecaller_results/recalibration/BaseCaller.json --calibration-json basecaller_results/Calibration.json --input-dir=sigproc_results --librarykey=TCAG --tfkey=ATCG --run-id=SEZXA --output-dir=basecaller_results --block-offset 0,0 --datasets=basecaller_results/datasets_pipeline.json --trim-adapter ATCACCGACTGCCCATAGAGAGGCTGAGACVN:5.0-13/e975447
@PG    ID:tmap    CL:mapall -n 1 -f /results/referenceLibrary/tmap-f3/e_coli_dh10b/e_coli_dh10b.fasta -r basecaller_results/rawlib.basecaller.bam -v -Y -u --prefix-exclude 5 --do-realign -o 2 stage1 map4    VN:5.0.13 (e975447) (201510291545)
@CO    {"BeadAdapters":{"Adapter_0":"ATCACCGACTGCCCATAGAGAGGCTGAGAC","MasterKey":"SEZXA","NumAdapters":1}}
@CO    {"MagicCode":"6d5b9d29ede5f176a4711d415d769108","MasterCol":0,"MasterKey":"SEZXA.block_X0_Y0","MasterRow":0,"SEZXA.block_X0_Y0":{"flowEnd":259,"flowSpan":130,"flowStart":0,"max_hp_calibrated":6,"min_hp_calibrated":4,"modelParameters":[{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9902219772338867,"paramB":0.01035221479833126,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9735603928565979,"paramB":0.01035221479833126,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9809113144874573,"paramB":-0.001820139237679541,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9809113144874573,"paramB":-0.001820139237679541,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.007941842079163,"paramB":-0.0003824764862656593,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.007941842079163,"paramB":-0.0003824764862656593,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9880667924880981,"paramB":-0.003570232540369034,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9843692183494568,"paramB":-0.003570232540369034,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9635631442070007,"paramB":0.03531987220048904,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9635631442070007,"paramB":0.03531987220048904,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9550077319145203,"paramB":0.01653131656348705,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9550077319145203,"paramB":0.01653131656348705,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9682118892669678,"paramB":0.02837859280407429,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9682118892669678,"paramB":0.02837859280407429,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9568409323692322,"paramB":0.005798361729830503,"refHP":4,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9568409323692322,"paramB":0.005798361729830503,"refHP":5,"xMax":49,"xMin":0,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9967536926269531,"paramB":0.006589567754417658,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9807540774345398,"paramB":0.006589567754417658,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9756661653518677,"paramB":-0.003973760176450014,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9756661653518677,"paramB":-0.003973760176450014,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.011394262313843,"paramB":-0.008048509247601032,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.011394262313843,"paramB":-0.008048509247601032,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9810723066329956,"paramB":-0.006564037408679724,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9775015115737915,"paramB":-0.006564037408679724,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9598845839500427,"paramB":0.02746859006583691,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9598845839500427,"paramB":0.02746859006583691,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9456416964530945,"paramB":0.0136557174846530,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9456416964530945,"paramB":0.0136557174846530,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9820154905319214,"paramB":0.01783179305493832,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9820154905319214,"paramB":0.01783179305493832,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9517754912376404,"paramB":0.003984427079558372,"refHP":4,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9517754912376404,"pa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Is there any tutorial or suggestions how to generate bam file with flow spaces to suites the tvc?

Note: i am using the latest version of TMAP and variant caller
alignment ion torrent variant caller • 3.5k views
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7.1 years ago
vmicrobio ▴ 290

I have the same problem Did you resolve it? Does anyone have a clue about this?

Thank you!
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7.1 years ago
Benn 8.3k

Did you contact life tech customer support? I don't think they scan this website for problems...
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thanks :) that's the next step I would have done

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Did you already get a respond from the customer support? I would also be interested in the answer, because I have the same problem...

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Yes, as expected it has been told that the Torrent Variant Caller requires FlowSpace data for working. In fastQ these data are not present, but in BAM files from the proton we can find them.

Of course they don't support modification of the code and discourage me to exclude flow order from IonTorrent data because of the potential noisy results.

So I have to find another way to get a vcf 'proton like', my aim being to compare Proton and Illumina data

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