FastaAlternateReferenceMaker not replacing sites in fasta file
Abnormal Results from FastaAlternateReferenceMaker:
FastaAlternateReferenceMaker appears to run fine (says it has completed with no errors, see log at the bottom) but I have seen no sign of any replacement of sites based on vcf file. Where are results for "--create-output-variant-md5" outputted to?
GATK and Java version numbers
gatk/4.4.0.0
java/jdk-17.0.2
Complete GATK Command
# IndexFeatureFile vcf
module load gatk/4.2.5.0
gatk IndexFeatureFile -I /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf
gatk FastaAlternateReferenceMaker \
-R /g/data/ht96/Mel_UQ/qf4/1_variant_call_pipeline/m_indica_alphonso_input_data/reference/peng_wang_alphonso_genome_multifasta_renamed.fa \
-V /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf \
-O /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/4_odorata_refgenome_test8.fasta \
--create-output-variant-md5 true --use-iupac-sample FUR9702A85
What previous methods you followed (Are you following the GATK Best Practices?)
## Creation of -V file
# Produce vcf with homozgyous genotypes only
module load bcftools/1.12
bcftools view --genotype hom /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/1_FUR9702A85_raw_variants.vcf \
-Oz -o /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/2_odorata/2_FUR9702A85_homosonly.vcf.gz
# Validate variants
gatk ValidateVariants \
-R /g/data/ht96/Mel_UQ/qf4/1_variant_call_pipeline/m_indica_alphonso_input_data/reference/peng_wang_alphonso_genome_multifasta_renamed.fa \
-V /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf \
--validation-type-to-exclude ALL
Relevant details about your analysis
I have created a vcf file with homozygous sites only for one genotype. I want to replace these alleles in the reference fasta.
Descriptions of what troubleshooting steps you have tried
- I have tried specifying a region, with and without "--use-iupac-sample" (there is only 1 individual in vcf file so shouldn't matter)
Illustrative examples of the issue you are seeing including an example of the results you expect
Chr10:660 should have switched G to an A based on -V file having the A genotype fixed in FUR9702A85
Output log:
(base) [wm6973@gadi-cpu-clx-0718 ~]$ gatk FastaAlternateReferenceMaker \
> -R /g/data/ht96/Mel_UQ/qf4/1_variant_call_pipeline/m_indica_alphonso_input_data/reference/peng_wang_alphonso_genome_multifasta_renamed.fa \
UQ/qf4/4_SIFT/derived_allele/1_odorataREF/4_odorata_refgenome_test8.fasta \
> -V /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf \
> -O /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/4_odorata_refgenome_test8.fasta \
> --create-output-variant-md5 true --use-iupac-sample FUR9702A85
Using GATK jar /apps/gatk/4.4.0.0/gatk-package-4.4.0.0-local.jar
Running:
java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /apps/gatk/4.4.0.0/gatk-package-4.4.0.0-local.jar FastaAlternateReferenceMaker -R /g/data/ht96/Mel_UQ/qf4/1_variant_call_pipeline/m_indica_alphonso_input_data/reference/peng_wang_alphonso_genome_multifasta_renamed.fa -V /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf -O /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/4_odorata_refgenome_test8.fasta --create-output-variant-md5 true --use-iupac-sample FUR9702A85
10:51:18.309 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/apps/gatk/4.4.0.0/gatk-package-4.4.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
10:51:18.334 INFO FastaAlternateReferenceMaker - ------------------------------------------------------------
10:51:18.337 INFO FastaAlternateReferenceMaker - The Genome Analysis Toolkit (GATK) v4.4.0.0
10:51:18.337 INFO FastaAlternateReferenceMaker - For support and documentation go to https://software.broadinstitute.org/gatk/
10:51:18.337 INFO FastaAlternateReferenceMaker - Executing as wm6973@gadi-cpu-clx-0718.gadi.nci.org.au on Linux v4.18.0-477.15.1.el8.nci.x86_64 amd64
10:51:18.337 INFO FastaAlternateReferenceMaker - Java runtime: OpenJDK 64-Bit Server VM v17.0.2+8-86
10:51:18.337 INFO FastaAlternateReferenceMaker - Start Date/Time: August 10, 2023 at 10:51:18 AM GMT+10:00
10:51:18.337 INFO FastaAlternateReferenceMaker - ------------------------------------------------------------
10:51:18.337 INFO FastaAlternateReferenceMaker - ------------------------------------------------------------
10:51:18.338 INFO FastaAlternateReferenceMaker - HTSJDK Version: 3.0.5
10:51:18.338 INFO FastaAlternateReferenceMaker - Picard Version: 3.0.0
10:51:18.338 INFO FastaAlternateReferenceMaker - Built for Spark Version: 3.3.1
10:51:18.338 INFO FastaAlternateReferenceMaker - HTSJDK Defaults.COMPRESSION_LEVEL : 2
10:51:18.338 INFO FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
10:51:18.338 INFO FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
10:51:18.339 INFO FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
10:51:18.339 INFO FastaAlternateReferenceMaker - Deflater: IntelDeflater
10:51:18.339 INFO FastaAlternateReferenceMaker - Inflater: IntelInflater
10:51:18.339 INFO FastaAlternateReferenceMaker - GCS max retries/reopens: 20
10:51:18.339 INFO FastaAlternateReferenceMaker - Requester pays: disabled
10:51:18.339 INFO FastaAlternateReferenceMaker - Initializing engine
10:51:18.445 INFO FeatureManager - Using codec VCFCodec to read file file:///g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/1_odorataREF/2_FUR9702A85_homosonly.vcf
10:51:19.037 INFO FastaAlternateReferenceMaker - Done initializing engine
10:51:19.046 INFO ProgressMeter - Starting traversal
10:51:19.046 INFO ProgressMeter - Current Locus Elapsed Minutes Bases Processed Bases/Minute
10:51:29.050 INFO ProgressMeter - chr10:14361000 0.2 14361000 86166000.0
10:51:39.046 INFO ProgressMeter - chr11:12059500 0.3 29712000 89136000.0
10:51:49.046 INFO ProgressMeter - chr12:10034926 0.5 44832000 89664000.0
10:51:59.046 INFO ProgressMeter - chr13:8878960 0.7 59706000 89559000.0
10:52:09.046 INFO ProgressMeter - chr14:8541966 0.8 74827000 89792400.0
10:52:19.046 INFO ProgressMeter - chr15:9042757 1.0 90138000 90138000.0
10:52:29.046 INFO ProgressMeter - chr16:10193797 1.2 106055000 90904285.7
10:52:39.046 INFO ProgressMeter - chr17:11847570 1.3 121526000 91144500.0
10:52:49.046 INFO ProgressMeter - chr19:10543 1.5 136567000 91044666.7
10:52:59.046 INFO ProgressMeter - chr1:2565477 1.7 152215000 91329000.0
10:53:09.046 INFO ProgressMeter - chr1:18088477 1.8 167738000 91493454.5
10:53:19.046 INFO ProgressMeter - chr20:3489877 2.0 182596000 91298000.0
10:53:29.046 INFO ProgressMeter - chr2:6843615 2.2 198244000 91497230.8
10:53:39.046 INFO ProgressMeter - chr2:22430615 2.3 213831000 91641857.1
10:53:49.046 INFO ProgressMeter - chr3:13148718 2.5 228947000 91578800.0
10:53:59.046 INFO ProgressMeter - chr4:5209325 2.7 244147000 91555125.0
10:54:09.046 INFO ProgressMeter - chr4:20725325 2.8 259663000 91645764.7
10:54:19.046 INFO ProgressMeter - chr5:14632825 3.0 275078000 91692666.7
10:54:29.046 INFO ProgressMeter - chr6:8855454 3.2 290384000 91700210.5
10:54:39.046 INFO ProgressMeter - chr7:5820494 3.3 306161000 91848300.0
10:54:49.046 INFO ProgressMeter - chr8:403374 3.5 321367000 91819142.9
10:54:59.046 INFO ProgressMeter - chr8:15976374 3.7 336940000 91892727.3
10:55:09.046 INFO ProgressMeter - chr9:13128905 3.8 352336000 91913739.1
10:55:19.046 INFO ProgressMeter - Un_0012:485236 4.0 369541000 92385250.0
10:55:29.046 INFO ProgressMeter - Un_0106:43121 4.2 389368000 93448320.0
10:55:30.443 INFO ProgressMeter - Un_0230:548 4.2 392983452 93791919.2
10:55:30.443 INFO ProgressMeter - Traversal complete. Processed 392983452 total bases in 4.2 minutes.
10:55:30.444 INFO FastaAlternateReferenceMaker - Shutting down engine
[August 10, 2023 at 10:55:30 AM GMT+10:00] org.broadinstitute.hellbender.tools.walkers.fasta.FastaAlternateReferenceMaker done. Elapsed time: 4.20 minutes.
Runtime.totalMemory()=1409286144
-
At the ValidateVariants step your command seems to disable all strict validation types. Is there a specific reason for this step? It may be possible that your vcf file and reference genome may have incompatibilites therefore tool cannot replace any bases.
Can you share the sequence dictionary of your reference fasta and VCF header?
-
I added "--validation-type-to-exclude ALL" as that was the suggested code to perform only VCF format tests.
Thanks for helping me!
.dict file for reference genome:
@HD VN:1.6
@SQ SN:chr10 LN:17652500 M5:9dec798a2fffecdca69924f771177028 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr11 LN:17144574 M5:725244fce90d72f68f1926706af5a09d UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr12 LN:16029966 M5:28ef0ca486442304ea64e1c1473431c6 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr13 LN:15457994 M5:5da43e7c39bd75acdd07080fdb0bd397 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr14 LN:14810209 M5:848ea963808a9a5321adaa027ef69a2e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr15 LN:14765960 M5:916e16691ca70fd69fbe8f3fe2842a3c UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr16 LN:13817227 M5:a4562e48ea3c60d43a543f2a40328265 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr17 LN:13506273 M5:993f9cf1857530afebf12bc5a1e7b1dd UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr18 LN:13371754 M5:36dddd446136d76554c9788a215cacbc UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr19 LN:13093066 M5:f1d38a44a8ea0c535a2d62fddb8b188b UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr1 LN:29456600 M5:f3351d34224f8b6fb30b101c33bbb1cc UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr20 LN:12294262 M5:ea60f186689b85cae78ca2e7bc804285 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr2 LN:24397897 M5:7d16d3d6d3695facfd38c549a331163e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr3 LN:23139393 M5:042b53863d5f7f27194070e5d7576c7f UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr4 LN:21507500 M5:99827b97e52bd39435c757fc63dee364 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr5 LN:21083371 M5:b6077cd6db0466053dd46911f6fffa42 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr6 LN:18811960 M5:d06d1e15cee677dfd1032c7557c68101 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr7 LN:20623120 M5:4860e42f1131bfdedd40b26b18172207 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr8 LN:18243469 M5:087279d4b8fe384921682f363fd48bba UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr9 LN:18233274 M5:a7a6e193757c73c75d275eabd23ccb4c UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chrM LN:871458 M5:ac7b4c25439eb4e11e03726c4134ad4f UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chrP LN:150346 M5:d6a0f36e21ad3a2e68a1814d874f360a UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0001 LN:1970699 M5:259b3b283352dba8cc970aa563e3e0f3 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0002 LN:1382144 M5:320e68138216ab8510d41b59ea170f2b UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0003 LN:1372446 M5:267626b3b31d2e7e30dd151bc8676b21 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0004 LN:1076389 M5:d7c81f310b0022a69dd1ae4921202a0e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0005 LN:872468 M5:96f7fd0621e15f3cd94c83f46b911953 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0006 LN:767780 M5:e004ddd089d69e45d63b6dd0e09a892b UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0007 LN:667392 M5:80360522b9a0ebe4d5a9dad7c24ec8f4 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0008 LN:627917 M5:f03fd9aa98ffb31782d46301822f4f2c UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0009 LN:621343 M5:82a68b471efb3bc1045106258fdf384c UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0010 LN:620923 M5:41e9900edcdd858aca05a81dac5f969f UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0011 LN:614090 M5:835f4df670c716e4b4f5d25f4d0d76d3 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0012 LN:558696 M5:f4cb5116a9d7d8fca7c2c84c3f64317e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0013 LN:529118 M5:73b7ababd27739002c088a6fb960108a UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0014 LN:504676 M5:8dd94638821b7443e6cfb7816203c424 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0015 LN:483988 M5:87cde649f6337a59177bca7563cda761 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0016 LN:470327 M5:08f50c927f1404dfd11f06852530ae91 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0017 LN:465424 M5:e2b554eefcaf0f09f50803e98e416bcc UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0018 LN:433226 M5:28761c477e6b092823defecfbb17c626 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0019 LN:428127 M5:14ab839121e8a38f9bed9d273d24c243 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0020 LN:427893 M5:760650a66868a16b7aefc011d52eff06 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0021 LN:406275 M5:d85b4717f5153803ab59e1242a62c53b UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0022 LN:395306 M5:5e36ec21a4b7570a3790847fc48abf5c UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0023 LN:394284 M5:44bd10c4333dc80549e428a9fed70bfe UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0024 LN:375682 M5:6494c43110b5e7a62eee9790fcf3597b UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0025 LN:358764 M5:b1d237e338a3b1bf9c072cd5b9f3b02e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0026 LN:350968 M5:2165eb8d528c46f5ff6a8d68df1a12f4 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0027 LN:338188 M5:87c4ab6c0ecbd823a060bcde7067d068 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0028 LN:336546 M5:9f56e9ebd708f878d0e151afcc1e5ccb UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0029 LN:311912 M5:20d8302a262e60a7c652f63b0b381833 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0030 LN:306605 M5:b2c00143e860e11a9f503a5a1d9dfdf6 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0031 LN:303935 M5:8fda820f3dc610ea7c386da1ee81088e UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0032 LN:301575 M5:35642220cc560865e887155fca007125 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0033 LN:274730 M5:648a53f439c8ad14649a7563ebd01dde UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0034 LN:274676 M5:6ae64a80b5358d84ee26b54bc5e4ac5f UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0035 LN:271678 M5:f0860ee8a72fdc455e9446e117335d96 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0036 LN:265704 M5:1c96e5e561159acd2b76e604669ec9ec UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0037 LN:256210 M5:5aa8081f6d7a3f782feb5ed382dcc12d UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0038 LN:255554 M5:6a24f6f4d9ed1a25c06e46e5b3f6296d UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0039 LN:247514 M5:1fc3cb27d326b5efb829804aab9f65b9 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0040 LN:244577 M5:38da75e1a50734c67aa4240198451a62 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
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@SQ SN:Un_0045 LN:232209 M5:a05f5a969dd7f893a3b016967c26a89a UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
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@SQ SN:Un_0124 LN:59801 M5:68f519e8a68b2d2e4fe2e17e4030bef5 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0125 LN:58552 M5:41b42f260c403b28e41f3c08083bdafd UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0126 LN:58488 M5:5da48c20c33b8ebf08d8b9685c73db82 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:Un_0127 LN:56628 M5:cb0bfcd306632b024955031b9b442d48 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
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@SQ SN:Un_0130 LN:49999 M5:41b7382b3394da1378067ecf12c4cb9f UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
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##bcftools_viewCommand=view --genotype hom -Oz -o /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/2_odorata/2_FUR9702A85_homosonly.vcf.gz /g/data/ht96/Mel_UQ/qf4/4_SIFT/derived_allele/2_odorata/1_FUR9702A85_raw_variants.vcf; Date=Fri May 5 09:29:10 2023
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT FUR9702A85 -
Sorry my post is still pending approval. Can you see it?
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Sorry, it got caught by our extremely overly aggressive filter and I didn't notice.
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Hello Melanie,
Thank you for providing the VCF header and the sequence dictionary. I have verified that sequences listed in the dictionary match those listen in the header in both length and name. I have also tried to replicate the reported behavior of FastaAlternateReferenceMaker, but without any success.
Before digging any deeper into the data itself, I want to verify that the tool is not making any changes to the new reference. When FastaAlternateReferenceMaker runs, it will generate a new sequence dictionary. Can you please share this sequence dictionary? If all the MD5s and lengths are the same, then we can assume no changes are propagating. If there are differences, then we need to figure out why they are not occurring at the expected loci. Thanks!
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Hi Giles,
Thank you for clarifying, we didn't know that the changes were only made to the .dict file and we were aligning the reads to the reference genome using BWA which doesn't use the .dict file.
I think mentioning that FastaAlternateReferenceMaker only modifies the .dict file would be helpful to add to the manual.
In saying that, I still don't think the .dict file is being modified correctly. The M5 in the .dict file is the same before and after running FastaAlternateReferenceMaker, the only thing that has changed in the .dict file is "SN:chr10" has change to "SN:1" .e.goriginal .dict
@SQ SN:chr10 LN:17652500 M5:9dec798a2fffecdca69924f771177028 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr11 LN:17144574 M5:725244fce90d72f68f1926706af5a09d UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr12 LN:16029966 M5:28ef0ca486442304ea64e1c1473431c6 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.fa
@SQ SN:chr13 LN:15457994 M5:5da43e7c39bd75acdd07080fdb0bd397 UR:file:/scratch/daff/mw/variant_calling_pipeline_gatk4/unified_workflow/reference/peng_wang_alphonso_genome_multifasta_renamed.faAfter FastaAlternateReferenceMaker:
@SQ SN:1 LN:17652500 M5:9dec798a2fffecdca69924f771177028
@SQ SN:2 LN:17144574 M5:725244fce90d72f68f1926706af5a09d
@SQ SN:3 LN:16029966 M5:28ef0ca486442304ea64e1c1473431c6
@SQ SN:4 LN:15457994 M5:5da43e7c39bd75acdd07080fdb0bd397
Where can I find the output for md5 after including "--create-output-variant-md5 true"? -
Hi Melanie,
Thank you for providing the before and after sequence dictionaries. Sorry for the confusion, but the changes are not exclusive to the sequence dictionary. I wanted to verify that the output reference was in fact the same as the input reference. Clearly, no changes to the reference are occurring.
I will circle back with the GATK engine team and see if they have any insight as to what is going on. I'm not sure where the --create-output-variant-md5 is stored, and will look into this as well.
Stay tuned.
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Hi Melanie,
We have a working hypothesis, but in order to verify, we will need to see a few examples from your VCF. Can you please share some examples? You can use this command will grab fifty sites:
head -1000 /path/to/vcf | tail -50
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Thanks for your help Giles! Here are the fifty sites:
chr10 2639 . G <NON_REF> . . END=2639 GT:DP:GQ:MIN_DP:PL 0/0:15:42:15:0,42,630
chr10 2640 . T <NON_REF> . . END=2640 GT:DP:GQ:MIN_DP:PL 0/0:15:36:15:0,36,540
chr10 2641 . T <NON_REF> . . END=2641 GT:DP:GQ:MIN_DP:PL 0/0:15:33:15:0,33,495
chr10 2642 . C <NON_REF> . . END=2642 GT:DP:GQ:MIN_DP:PL 0/0:16:36:16:0,36,540
chr10 2644 . A <NON_REF> . . END=2647 GT:DP:GQ:MIN_DP:PL 0/0:17:39:17:0,39,585
chr10 2648 . C <NON_REF> . . END=2648 GT:DP:GQ:MIN_DP:PL 0/0:17:36:17:0,36,629
chr10 2649 . A <NON_REF> . . END=2655 GT:DP:GQ:MIN_DP:PL 0/0:13:39:13:0,39,478
chr10 2656 . T <NON_REF> . . END=2661 GT:DP:GQ:MIN_DP:PL 0/0:14:42:14:0,42,520
chr10 2662 . C <NON_REF> . . END=2662 GT:DP:GQ:MIN_DP:PL 0/0:15:45:15:0,45,576
chr10 2663 . T <NON_REF> . . END=2664 GT:DP:GQ:MIN_DP:PL 0/0:15:29:15:0,29,469
chr10 2665 . T <NON_REF> . . END=2665 GT:DP:GQ:MIN_DP:PL 0/0:15:30:15:0,30,569
chr10 2666 . T <NON_REF> . . END=2667 GT:DP:GQ:MIN_DP:PL 0/0:16:42:16:0,42,630
chr10 2668 . T <NON_REF> . . END=2668 GT:DP:GQ:MIN_DP:PL 0/0:16:33:16:0,33,577
chr10 2669 . A <NON_REF> . . END=2671 GT:DP:GQ:MIN_DP:PL 0/0:16:42:16:0,42,630
chr10 2672 . T <NON_REF> . . END=2678 GT:DP:GQ:MIN_DP:PL 0/0:17:48:16:0,48,604
chr10 2679 . G A,<NON_REF> 706.03 . DP=14;ExcessHet=3.0103;MLEAC=2,0;MLEAF=1,0;RAW_MQandDP=50400,14 GT:AD:DP:GQ:PGT:PID:PL:PS:SB 1|1:0,14,0:14:48:0|1:2679_G_A:720,48,0,720,48,720:2679:0,0,5,9
chr10 2680 . A <NON_REF> . . END=2683 GT:DP:GQ:MIN_DP:PL 0/0:16:48:16:0,48,608
chr10 2684 . G <NON_REF> . . END=2684 GT:DP:GQ:MIN_DP:PL 0/0:16:33:16:0,33,577
chr10 2685 . C <NON_REF> . . END=2707 GT:DP:GQ:MIN_DP:PL 0/0:16:48:16:0,48,596
chr10 2708 . A <NON_REF> . . END=2709 GT:DP:GQ:MIN_DP:PL 0/0:18:51:18:0,51,765
chr10 2711 . A <NON_REF> . . END=2712 GT:DP:GQ:MIN_DP:PL 0/0:18:51:18:0,51,765
chr10 2713 . A <NON_REF> . . END=2727 GT:DP:GQ:MIN_DP:PL 0/0:20:60:20:0,60,695
chr10 2728 . T <NON_REF> . . END=2732 GT:DP:GQ:MIN_DP:PL 0/0:20:57:20:0,57,855
chr10 2733 . A <NON_REF> . . END=2739 GT:DP:GQ:MIN_DP:PL 0/0:23:63:23:0,63,945
chr10 2741 . T <NON_REF> . . END=2751 GT:DP:GQ:MIN_DP:PL 0/0:23:66:22:0,66,803
chr10 2752 . A <NON_REF> . . END=2760 GT:DP:GQ:MIN_DP:PL 0/0:24:72:24:0,72,870
chr10 2762 . T <NON_REF> . . END=2781 GT:DP:GQ:MIN_DP:PL 0/0:27:72:25:0,72,933
chr10 2782 . C <NON_REF> . . END=2784 GT:DP:GQ:MIN_DP:PL 0/0:25:69:24:0,69,1035
chr10 2786 . C <NON_REF> . . END=2787 GT:DP:GQ:MIN_DP:PL 0/0:24:69:24:0,69,1035
chr10 2788 . T <NON_REF> . . END=2806 GT:DP:GQ:MIN_DP:PL 0/0:26:72:24:0,72,910
chr10 2807 . T <NON_REF> . . END=2807 GT:DP:GQ:MIN_DP:PL 0/0:25:69:25:0,69,1035
chr10 2808 . C <NON_REF> . . END=2811 GT:DP:GQ:MIN_DP:PL 0/0:26:72:25:0,72,1080
chr10 2812 . A <NON_REF> . . END=2813 GT:DP:GQ:MIN_DP:PL 0/0:25:66:25:0,66,990
chr10 2814 . C <NON_REF> . . END=2816 GT:DP:GQ:MIN_DP:PL 0/0:27:72:27:0,72,1080
chr10 2817 . A <NON_REF> . . END=2819 GT:DP:GQ:MIN_DP:PL 0/0:27:69:27:0,69,1035
chr10 2820 . C <NON_REF> . . END=2823 GT:DP:GQ:MIN_DP:PL 0/0:28:75:27:0,75,1125
chr10 2825 . G <NON_REF> . . END=2837 GT:DP:GQ:MIN_DP:PL 0/0:26:75:25:0,75,960
chr10 2838 . A <NON_REF> . . END=2847 GT:DP:GQ:MIN_DP:PL 0/0:28:81:27:0,81,1043
chr10 2848 . A <NON_REF> . . END=2853 GT:DP:GQ:MIN_DP:PL 0/0:28:75:28:0,75,1125
chr10 2854 . A <NON_REF> . . END=2854 GT:DP:GQ:MIN_DP:PL 0/0:28:69:28:0,69,1035
chr10 2855 . G <NON_REF> . . END=2861 GT:DP:GQ:MIN_DP:PL 0/0:28:72:28:0,72,1080
chr10 2863 . G <NON_REF> . . END=2872 GT:DP:GQ:MIN_DP:PL 0/0:25:72:24:0,72,965
chr10 2873 . G <NON_REF> . . END=2886 GT:DP:GQ:MIN_DP:PL 0/0:26:63:24:0,63,945
chr10 2887 . C <NON_REF> . . END=2887 GT:DP:GQ:MIN_DP:PL 0/0:25:72:25:0,72,1080
chr10 2888 . A <NON_REF> . . END=2894 GT:DP:GQ:MIN_DP:PL 0/0:25:69:24:0,69,1035
chr10 2897 . G <NON_REF> . . END=2903 GT:DP:GQ:MIN_DP:PL 0/0:26:69:24:0,69,1035
chr10 2904 . C <NON_REF> . . END=2907 GT:DP:GQ:MIN_DP:PL 0/0:25:72:25:0,72,1080
chr10 2908 . T <NON_REF> . . END=2922 GT:DP:GQ:MIN_DP:PL 0/0:24:63:22:0,63,945
chr10 2923 . T <NON_REF> . . END=2923 GT:DP:GQ:MIN_DP:PL 0/0:23:54:23:0,54,845
chr10 2924 . G <NON_REF> . . END=2928 GT:DP:GQ:MIN_DP:PL 0/0:22:66:22:0,66,808 -
Hi Melanie,
We believe the problem is that FastaAlternateReferenceMaker is not compatible with GVCF files. You can convert GVCFs to VCFs with the following command:
gatk --java-options "-Xmx4g" GenotypeGVCFs \
-R original_reference.fasta \
-V input.g.vcf.gz \
-O output.vcf.gzCan you please try converting your GVCF to a VCF, and then run FastaAlternateReferenceMaker on the genotyped VCF?
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That worked!!!! It modified the fasta file based on the vcf!
Thanks so much for your help Giles!!
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