Hi there, 

I am trying to output a multisample VCF from a genomicsDB.

The GenomicsDB was created with 108 g.vcf files, in turn generated using Clara Parabrick's accelerated germline pipeline (v4.0.1). The command used is:

#to make the DB

gatk --java-options -Xmx150G GenomicsDBImport --genomicsdb-workspace-path germline_0323 --sample-name-map map.map -L intervals.list --reader-threads 20 --max-num-intervals-to-import-in-parallel 3

#to make final VCF

gatk GenotypeGVCFs -R ../ref/Genome.fasta -V gendb://germline_0323 -O output.vcf --max-alternate-alleles 5

The bottom ~100 lines of the log (it's very long) is below. When I try and run GenotypeGVCFs on a merged GVCF (the old way) the same error happens. In a very close position (CM023248:28015217  vs CM023248:28016217). 

I have searched the genome for non-IUPAC characters and found none. Similarly, when I manually examine the few 1000BP in this region, all of the characters are ACTG.

Do you think it may be to do with the warnings about there being too many alternative alleles?

GATK version is 4.3.0

Any help would be greatly appreciated - thank you!

Tristan

Log here:

Sample/Callset C1aSud049( TileDB row idx 32) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud050( TileDB row idx 33) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud051( TileDB row idx 34) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud052( TileDB row idx 35) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud053( TileDB row idx 36) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
/Callset C1aSud054( TileDB row idx 37) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud055( TileDB row idx 38) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud056( TileDB row idx 39) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud058( TileDB row idx 40) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud059( TileDB row idx 41) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud060( TileDB row idx 42) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud061( TileDB row idx 43) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud063( TileDB row idx 44) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud064( TileDB row idx 45) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud065( TileDB row idx 46) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
Sample/Callset C1aSud066( TileDB row idx 47) at Chromosome CM023248 position 26846163 (TileDB column 26846162) has too many genotypes in the combined VCF record : 1081 : current limit : 1024 (num_alleles, ploidy) = (46, 2). Fields, such as  PL, with length equal to the number of genotypes will NOT be added   for this sample for this location.
13:06:20.682 WARN  MinimalGenotypingEngine - No genotype contained sufficient data to recalculate site and allele qualities. Site will be skipped at location CM023248:26846163
13:06:30.188 INFO  ProgressMeter -    CM023248:26860204            216.5              26364000         121768.9
13:06:40.231 INFO  ProgressMeter -    CM023248:26877204            216.7              26381000         121753.3
13:06:50.552 INFO  ProgressMeter -    CM023248:26896214            216.8              26400000         121744.3
13:07:00.684 INFO  ProgressMeter -    CM023248:26917218            217.0              26421000         121746.4
13:07:10.845 INFO  ProgressMeter -    CM023248:26933223            217.2              26437000         121725.1
13:07:21.114 INFO  ProgressMeter -    CM023248:26951224            217.4              26455000         121712.1
13:07:31.557 INFO  ProgressMeter -    CM023248:26970260            217.5              26474000         121702.0
13:07:41.848 INFO  ProgressMeter -    CM023248:26990262            217.7              26494000         121698.0
13:07:52.202 INFO  ProgressMeter -    CM023248:27013457            217.9              26517000         121707.2
13:08:02.461 INFO  ProgressMeter -    CM023248:27031467            218.0              26535000         121694.3
13:08:12.598 INFO  ProgressMeter -    CM023248:27048469            218.2              26552000         121678.0
13:08:22.884 INFO  ProgressMeter -    CM023248:27066497            218.4              26570000         121664.9
13:08:33.266 INFO  ProgressMeter -    CM023248:27090947            218.6              26586000         121641.8
13:08:43.548 INFO  ProgressMeter -    CM023248:27110991            218.7              26606000         121637.9
13:08:53.601 INFO  ProgressMeter -    CM023248:27127006            218.9              26622000         121617.9
13:09:04.899 INFO  ProgressMeter -    CM023248:27144047            219.1              26639000         121591.0
13:09:14.950 INFO  ProgressMeter -    CM023248:27166064            219.3              26661000         121598.4
13:09:25.449 INFO  ProgressMeter -    CM023248:27195553            219.4              26684000         121606.3
13:09:35.923 INFO  ProgressMeter -    CM023248:27212205            219.6              26700000         121582.4
13:09:46.055 INFO  ProgressMeter -    CM023248:27227250            219.8              26715000         121557.3
13:09:56.326 INFO  ProgressMeter -    CM023248:27244563            219.9              26732000         121540.0
13:10:06.726 INFO  ProgressMeter -    CM023248:27271173            220.1              26752000         121535.1
13:10:17.242 INFO  ProgressMeter -    CM023248:27291173            220.3              26772000         121529.2
13:10:27.372 INFO  ProgressMeter -    CM023248:27310181            220.5              26791000         121522.3
13:10:37.550 INFO  ProgressMeter -    CM023248:27329181            220.6              26810000         121515.0
13:10:48.548 INFO  ProgressMeter -    CM023248:27352181            220.8              26833000         121518.3
13:10:58.661 INFO  ProgressMeter -    CM023248:27371181            221.0              26852000         121511.6
13:11:08.741 INFO  ProgressMeter -    CM023248:27392212            221.2              26873000         121514.2
13:11:18.813 INFO  ProgressMeter -    CM023248:27415019            221.3              26894000         121517.0
13:11:29.514 INFO  ProgressMeter -    CM023248:27433236            221.5              26912000         121500.4
13:11:39.693 INFO  ProgressMeter -    CM023248:27455236            221.7              26934000         121506.6
13:11:49.712 INFO  ProgressMeter -    CM023248:27476473            221.8              26953000         121500.8
13:11:59.984 INFO  ProgressMeter -    CM023248:27492473            222.0              26969000         121479.2
13:12:10.226 INFO  ProgressMeter -    CM023248:27515032            222.2              26991000         121484.9
13:12:20.498 INFO  ProgressMeter -    CM023248:27533033            222.3              27009000         121472.3
13:12:30.953 INFO  ProgressMeter -    CM023248:27551033            222.5              27027000         121458.1
13:12:41.274 INFO  ProgressMeter -    CM023248:27570034            222.7              27046000         121449.6
13:12:51.577 INFO  ProgressMeter -    CM023248:27586138            222.9              27062000         121427.8
13:13:01.606 INFO  ProgressMeter -    CM023248:27601141            223.0              27077000         121404.0
13:13:12.173 INFO  ProgressMeter -    CM023248:27620141            223.2              27096000         121393.4
13:13:22.340 INFO  ProgressMeter -    CM023248:27640141            223.4              27116000         121390.8
13:13:32.837 INFO  ProgressMeter -    CM023248:27656141            223.6              27132000         121367.4
13:13:43.144 INFO  ProgressMeter -    CM023248:27676141            223.7              27152000         121363.6
13:13:53.579 INFO  ProgressMeter -    CM023248:27696141            223.9              27172000         121358.7
13:14:03.944 INFO  ProgressMeter -    CM023248:27713150            224.1              27189000         121341.0
13:14:14.377 INFO  ProgressMeter -    CM023248:27730152            224.2              27206000         121322.7
13:14:25.005 INFO  ProgressMeter -    CM023248:27749158            224.4              27225000         121311.6
13:14:35.210 INFO  ProgressMeter -    CM023248:27771158            224.6              27247000         121317.7
Chromosome CM023248 position 27776521 (TileDB column 27776520) has too many alleles in the combined VCF record : 54 : current limit : 50. Fields, such as  PL, with length equal to the number of genotypes will NOT be added for this location.
13:14:39.313 WARN  MinimalGenotypingEngine - Attempting to genotype more than 50 alleles. Site will be skipped at location CM023248:27776521
13:14:45.456 INFO  ProgressMeter -    CM023248:27787160            224.8              27263000         121296.7
13:14:55.585 INFO  ProgressMeter -    CM023248:27810794            224.9              27283000         121294.6
13:15:05.870 INFO  ProgressMeter -    CM023248:27831107            225.1              27303000         121291.0
13:15:15.968 INFO  ProgressMeter -    CM023248:27852122            225.3              27324000         121293.6
13:15:26.346 INFO  ProgressMeter -    CM023248:27871282            225.4              27343000         121284.9
13:15:36.732 INFO  ProgressMeter -    CM023248:27893284            225.6              27365000         121289.3
13:15:47.359 INFO  ProgressMeter -    CM023248:27911284            225.8              27383000         121273.9
13:15:57.738 INFO  ProgressMeter -    CM023248:27928284            226.0              27400000         121256.3
13:16:08.138 INFO  ProgressMeter -    CM023248:27947360            226.1              27419000         121247.4
Chromosome CM023248 position 27954185 (TileDB column 27954184) has too many alleles in the combined VCF record : 85 : current limit : 50. Fields, such as  PL, with length equal to the number of genotypes will NOT be added for this location.
13:16:11.766 WARN  MinimalGenotypingEngine - Attempting to genotype more than 50 alleles. Site will be skipped at location CM023248:27954185
13:16:18.678 INFO  ProgressMeter -    CM023248:27968185            226.3              27439000         121241.6
13:16:29.128 INFO  ProgressMeter -    CM023248:27983185            226.5              27454000         121214.6
13:16:39.280 INFO  ProgressMeter -    CM023248:27999202            226.7              27470000         121194.7
13:16:49.283 INFO  ProgressMeter -    CM023248:28015217            226.8              27486000         121176.2
13:16:57.733 INFO  GenotypeGVCFs - Shutting down engine
GENOMICSDB_TIMER,GenomicsDB iterator next() timer,Wall-clock time(s),3971.471023722291,Cpu time(s),3943.851816477288
[22 March 2023 13:16:58 GMT] org.broadinstitute.hellbender.tools.walkers.GenotypeGVCFs done. Elapsed time: 227.36 minutes.
Runtime.totalMemory()=2109734912
***********************************************************************
A USER ERROR has occurred: Bad input: We encountered a non-standard non-IUPAC base in the provided input sequence: '0'