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,"paramB":0.003984427079558372,"refHP":5,"xMax":99,"xMin":50,"yMax":49,"yMin":0},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9738200306892395,"paramB":0.002562004607170820,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9681893587112427,"paramB":0.002562004607170820,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9714478254318237,"paramB":0.004633536096662283,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9714478254318237,"paramB":0.004633536096662283,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.020444154739380,"paramB":-0.0009156712912954390,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.020444154739380,"paramB":-0.0009156712912954390,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9661490917205811,"paramB":-0.005131537560373545,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9659626483917236,"paramB":-0.005131537560373545,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9532436728477478,"paramB":0.02550310641527176,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9532436728477478,"paramB":0.02550310641527176,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9405366778373718,"paramB":0.02434134855866432,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9405366778373718,"paramB":0.02434134855866432,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9701055288314819,"paramB":0.03062361106276512,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9701055288314819,"paramB":0.03062361106276512,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9403199553489685,"paramB":0.006568837910890579,"refHP":4,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9403199553489685,"paramB":0.006568837910890579,"refHP":5,"xMax":49,"xMin":0,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9858608245849609,"paramB":0.007273687515407801,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":129,"flowStart":0,"paramA":0.9729442000389099,"paramB":0.007273687515407801,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9865635037422180,"paramB":-0.001045344513840973,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":129,"flowStart":0,"paramA":0.9865635037422180,"paramB":-0.001045344513840973,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.008965015411377,"paramB":-0.002915073186159134,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":129,"flowStart":0,"paramA":1.008965015411377,"paramB":-0.002915073186159134,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9763863086700439,"paramB":-0.002955069765448570,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":129,"flowStart":0,"paramA":0.9798527359962463,"paramB":-0.002955069765448570,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9750684499740601,"paramB":0.03322410583496094,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":65,"flowEnd":259,"flowStart":130,"paramA":0.9750684499740601,"paramB":0.03322410583496094,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9596220850944519,"paramB":0.01509375963360071,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":67,"flowEnd":259,"flowStart":130,"paramA":0.9596220850944519,"paramB":0.01509375963360071,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9796839952468872,"paramB":0.02462446317076683,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":71,"flowEnd":259,"flowStart":130,"paramA":0.9796839952468872,"paramB":0.02462446317076683,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9565287828445435,"paramB":0.009266614913940430,"refHP":4,"xMax":99,"xMin":50,"yMax":99,"yMin":50},{"flowBase":84,"flowEnd":259,"flowStart":130,"paramA":0.9565287828445435,"paramB":0.009266614913940430,"refHP":5,"xMax":99,"xMin":50,"yMax":99,"yMin":50}],"xMax":99,"xMin":0,"xSpan":50,"yMax":99,"yMin":0,"ySpan":50}}

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