GATK IndelRealigner error

[kga1978]

Hi All,
I am trying to perform a local realignment of some BAM generated with Novoalign. I run the following commands:

Command 1:
novoalign -f reads.fastq.gz -c 2 -d Mosaik/reference -o SAM 2> reads.novoalign_logS0.txt | samtools view -S -b -q 1 - | samtools sort - reads

Command 2:
java -Xmx2g -jar /usr/local/bin/picard/SortSam.jar I=reads.bam O=readssorted.bam SO=coordinate

Command 3:
java -Xmx2g -jar /usr/local/bin/gatk/GenomeAnalysisTK.jar -I readssorted.bam -R Mosaik/reference.fasta -T RealignerTargetCreator -o forIndelRealigner.intervals

Command 4:
java -Xmx2g -jar /usr/local/bin/gatk/GenomeAnalysisTK.jar -I readssorted.bam -R Mosaik/reference.fasta -T IndelRealigner --targetIntervals forIndelRealigner.intervals -o realignedBam.bam

The RealignerTargetCreator (3) finishes successfully and creates the required file:

LASV-reference:52-53
LASV-reference:305-339
LASV-reference:439-519

However, when I run the last command (4) - IndelRealigner, I get the following error:

##### ERROR MESSAGE: File associated with name forIndelRealigner.intervals is malformed: Interval file could not be parsed in any supported format. caused by Failed to parse Genome Location string: LASV-reference:52-53

Any idea what might be the problem here? I have tried various things, but I always fail here.

My reference.dict looks like this:
@HD VN:1.0 SO:unsorted
@SQ SN:LASV-reference LN:3402 UR:file:/Users/kga/Desktop/Mosaik/reference.fasta M5:8a4c76005c28bef3f2775dbf6ffa2062

reference.fasta.fai:
LASV-reference 3402 89 3402 3403


Thanks very much,
Kristian

[Heisman]

Could you maybe just post the first couple dozen lines of the various files used (or if a bunch of lines look similar just a representative line)?

[kga1978]

Sure thing:

Reference:
>LASV-reference
GCGCACAGTGGATCCTAGGCATTTTTGGTTGCGCAATTCAAGTGTCCTATTTAAAATGGGACAAATAGTGACATTCTTCCAGGAAGTGCCTCATGTAATAGAAGAGGTGATGAACATTGTTCTCATTGCACTGTCTGTACTAGCAGTGCTGAAAGGTCTGTACAATTTTGCAACGTGTGGCCTTGTTGGTTTGGTCACTTTCCTCCTGTTGTGTGGTAGGTCTTGCACAACCAGTCTTTATAAAGGGGTTTATGAGCTTCAGACTCTGGAACTAAACATGGAGACACTCAATATGACCATGCCTCTCTCCTGCACAAAGAACAACAGTCATCATTATATAATGGTGGGCAATGAGACAGGACTAGAACTGACCTTGACCAACACGAGCATTATTAATCACAAATTTTGCAATCTGTCTGATGCCCACAAAAAGAACCTCTATGACCACGCTCTTATGAGCATAATCTCAACTTTCCACTTGTCCATCCCCAACTTCAATCAGTATGAGGCAATGAGCTGCGATTTTAATGGGGGAAA

Sorted BAM file:
ILLUMINA_0142:3:1108:12467:139455#TGACCA/1 0 LASV-reference 2892 20 1S51M * 0 0 GTCTTTGGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACT Z_^cc`ce^aeegedghe_gggcfdhdhhaX^dfghfhhhdhdedg_dfdgh RG:Z:ZGO3HPVJRLW NM:i:2 MD:Z:24T1G24 ZA:Z:<@;0;0;;1;;>
ILLUMINA_0142:3:1104:8199:92212#TGACCA/1 0 LASV-reference 2893 19 52M * 0 0 CTTTGGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACTCA ^__cc``Yaa^b`beefhehhddf]dfgfhhRabcdbg`fffbcffghhfhf RG:Z:ZGO3HPVJRLW NM:i:3 MD:Z:23T1G24T1 ZA:Z:<@;0;0;;1;;>
ILLUMINA_0142:3:1108:20971:8153#TGACCA/1 16 LASV-reference 2893 19 52M * 0 0 CTTTGGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACTCA caQRccbeefcecb^PI[dXeb[X`hefdXbSSgbSd_`Qb[eecSc``__^ RG:Z:ZGO3HPVJRLW NM:i:3 MD:Z:23T1G24T1 ZA:Z:<@;0;0;;1;;>
ILLUMINA_0142:3:2102:12125:81885#TGACCA/1 16 LASV-reference 2894 18 51M1S * 0 0 TTTGGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACTCAG Z_f^fd_abhgbd`cfbdbbYJ`JRe\gebXec`e`Yb[cbabba\`cc__\ RG:Z:ZGO3HPVJRLW NM:i:3 MD:Z:22T1G24T1 ZA:Z:<@;0;0;;1;;>
ILLUMINA_0142:3:1208:5666:190436#TGACCA/1 16 LASV-reference 2895 20 52M * 0 0 TTGGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACTCAAT c]dhee^ee^^Hehfe_deebdeZeehebgd_gafabQJJeeeeccccc___ RG:Z:ZGO3HPVJRLW NM:i:3 MD:Z:21T1G24T3 ZA:Z:<@;0;0;;1;;>
ILLUMINA_0142:3:1204:18832:77734#TGACCA/1 0 LASV-reference 2897 19 49M * 0 0 GGTCAAGTTGCTGTGAGCTCAAGTTGCCCATATAGACACCTGCACTCAA abaeeeecggfggghhgfhihiiggiiiiiiiiiihiiiiiiiihiiih RG:Z:ZGO3HPVJRLW NM:i:3 MD:Z:19T1G24T2 ZA:Z:<@;0;0;;1;;>

The .intervals, .fai and .dict files are exactly as described above - no further text in those.

Thanks very much

[kga1978]

Anybody any thoughts? This is driving me nuts and SRMA doesn't appear to be working either (separate post)

Thanks in advance.

[swbarnes2]

Does your fasta file really say "reference", and not "LASV-reference"?

[kga1978]

Sorry, that is my bad - I tried to make another reference with just the word 'reference' - but the one I have been using correctly says 'LASV-reference' - I have corrected the typo.

[Bukowski]

You do know you can get in touch directly with the GATK team here:

http://getsatisfaction.com/gsa

They're very responsive to questions.

[JLand52]

GATK is picky about the file name. Try changing the extension to ".interval_list"