All intervals were filtered in Filterintervals
I am working on the non-human primates' taste receptor analysis and trying to get the ploidy for each gene. When I ran the gatk germline pipeline, I set the bin length 0 in PreprocessIntervals and gave a bed file for all genes that I mapped, all the intervals were removed after I ran the FilterIntervals. Below is the log, plsease help me to solve the problem. Thanks!
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 /usr/local/bin/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar FilterIntervals -L pre_Cmit.interval_list --annotated-intervals ann_Cmit1.tsv --interval-merging-rule OVERLAPPING_ONLY -O Cmit_filter.interval_list
12:45:49.902 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/usr/local/bin/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
May 24, 2022 12:45:50 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
12:45:50.067 INFO FilterIntervals - ------------------------------------------------------------
12:45:50.067 INFO FilterIntervals - The Genome Analysis Toolkit (GATK) v4.2.5.0
12:45:50.067 INFO FilterIntervals - For support and documentation go to https://software.broadinstitute.org/gatk/
12:45:50.068 INFO FilterIntervals - Executing as visual-ngs@visualngs on Linux v5.4.0-100-generic amd64
12:45:50.068 INFO FilterIntervals - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_171-b11
12:45:50.068 INFO FilterIntervals - Start Date/Time: May 24, 2022 12:45:49 PM JST
12:45:50.068 INFO FilterIntervals - ------------------------------------------------------------
12:45:50.068 INFO FilterIntervals - ------------------------------------------------------------
12:45:50.069 INFO FilterIntervals - HTSJDK Version: 2.24.1
12:45:50.069 INFO FilterIntervals - Picard Version: 2.25.4
12:45:50.069 INFO FilterIntervals - Built for Spark Version: 2.4.5
12:45:50.069 INFO FilterIntervals - HTSJDK Defaults.COMPRESSION_LEVEL : 2
12:45:50.069 INFO FilterIntervals - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
12:45:50.069 INFO FilterIntervals - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
12:45:50.069 INFO FilterIntervals - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
12:45:50.069 INFO FilterIntervals - Deflater: IntelDeflater
12:45:50.069 INFO FilterIntervals - Inflater: IntelInflater
12:45:50.069 INFO FilterIntervals - GCS max retries/reopens: 20
12:45:50.069 INFO FilterIntervals - Requester pays: disabled
12:45:50.070 INFO FilterIntervals - Initializing engine
12:45:50.070 INFO FilterIntervals - Done initializing engine
12:45:50.554 INFO FeatureManager - Using codec IntervalListCodec to read file file:///home/visual-ngs/Desktop/data2/Min/OWM/Cmit/3rd_mapping4_5/Cmit/CNV_test/bam1/pre_Cmit.interval_list
12:45:50.597 INFO IntervalArgumentCollection - Processing 276666 bp from intervals
12:45:50.600 INFO FilterIntervals - After interval resolution, 116 intervals remain...
12:45:50.600 INFO FilterIntervals - After interval intersection, 116 intervals remain...
12:45:50.604 INFO FilterIntervals - Applying annotation-based filters...
12:45:50.611 INFO FilterIntervals - After applying GC-content filter (intervals with values outside of [0.1, 0.9] fail), 116 / 116 intervals remain...
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig Mmu_TAS2R15P_0-916 was left with a single interval that was filtered out.
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig NR19_Csa_0-1534 was left with a single interval that was filtered out.
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig NR11_Csa_0-1293 was left with a single interval that was filtered out.
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig NR49_Csa_0-5182 was left with a single interval that was filtered out.
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig 18_Ptr_TAS2R30_1001-1960_0-961 was left with a single interval that was filtered out.
12:45:50.618 WARN FilterIntervals - After applying provided filters, contig NR20_Csa_0-4174 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R411_0-930 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR17_Can1_0-3695 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR85_Csa_0-2181 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR76_Csa_0-3167 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR81_Csa_0-2076 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR15_Csa_0-1592 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR41_Csa_0-2536 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR57_Csa_0-1630 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR39_Csa_0-5233 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR77_Csa_0-3037 was left with a single interval that was filtered out.
12:45:50.619 WARN FilterIntervals - After applying provided filters, contig NR7_Csa_0-1679 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR62_Csa_0-2052 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR84_Csa_0-2160 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR67_Csa_0-2168 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR82_Csa_0-1041 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR16_Csa_0-843 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R419_0-927 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR60_Csa_0-1171 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R401P_0-903 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR45_Csa_0-12059 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR75_Csa_0-12020 was left with a single interval that was filtered out.
12:45:50.620 WARN FilterIntervals - After applying provided filters, contig NR9_Mmu_0-1247 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR56_Csa_0-2636 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR43_Can_0-5707 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR66_Csa_0-1379 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR6_Csa_0-1174 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR50_Csa_0-2227 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR46_Csa_0-6275 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR72_Csa_0-5979 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR8_Csa_0-3534 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR53_Csa_0-1319 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR35_Csa_0-1471 was left with a single interval that was filtered out.
12:45:50.621 WARN FilterIntervals - After applying provided filters, contig NR55_Csa_0-2249 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR17_Csa2_0-2724 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR29_Csa_0-1094 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR1_Csa_0-1989 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR23_Can_0-5222 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR54_Csa_0-1056 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR48_Csa_0-2243 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR5_Csa_0-6046 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR59_Csa_0-1296 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR68_Csa_0-1269 was left with a single interval that was filtered out.
12:45:50.622 WARN FilterIntervals - After applying provided filters, contig NR51_Csa_0-3825 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR79_Csa_0-1125 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR47_Csa_0-2819 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR69_Csa_0-1522 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR28_Csa_0-2592 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR13_Csa_0-2482 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR44_Csa_0-2082 was left with a single interval that was filtered out.
12:45:50.623 WARN FilterIntervals - After applying provided filters, contig NR3_Csa_0-1388 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR30_Csa_0-1242 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR63_Csa_0-1756 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR58_Csa_0-1680 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR37_Csa_0-1781 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR74_Csa_0-13008 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R62_0-915 was left with a single interval that was filtered out.
12:45:50.624 WARN FilterIntervals - After applying provided filters, contig NR78_Csa_0-1199 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR73_Csa_0-15142 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR36_Csa_0-2822 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR52_Csa_0-2359 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR38_Csa_0-1439 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR40_Csa_0-1158 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig NR4_Csa_0-1155 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig 1_Cat_TAS2R416_1001-1891_0-891 was left with a single interval that was filtered out.
12:45:50.625 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R39_0-967 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R40_0-972 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig NR33_Csa_0-5296 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig NR34_Csa_0-1305 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig NR65_Csa_0-2316 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R60_0-957 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig NR12_Csa_0-1161 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig NR80_Csa_0-1036 was left with a single interval that was filtered out.
12:45:50.626 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R3_0-951 was left with a single interval that was filtered out.
12:45:50.627 WARN FilterIntervals - After applying provided filters, contig NR32_Csa_0-999 was left with a single interval that was filtered out.
12:45:50.627 WARN FilterIntervals - After applying provided filters, contig NR64_Csa_0-11267 was left with a single interval that was filtered out.
12:45:50.628 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R5_0-900 was left with a single interval that was filtered out.
12:45:50.628 WARN FilterIntervals - After applying provided filters, contig NR2_Csa_0-6716 was left with a single interval that was filtered out.
12:45:50.628 WARN FilterIntervals - After applying provided filters, contig NR83_Csa_0-1547 was left with a single interval that was filtered out.
12:45:50.628 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R1_0-900 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R4_0-900 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R16_0-876 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R41_0-924 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig NR31_Csa_0-4427 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R38_0-1002 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig NR10_Csa_0-1153 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig NR25_Csa_0-2339 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig NR22_Csa_0-4958 was left with a single interval that was filtered out.
12:45:50.629 WARN FilterIntervals - After applying provided filters, contig NR21_Csa_0-1302 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig NR26_Csa_0-1132 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig 535_Rro_TAS2R417_0-927 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig NR42_Csa_0-1785 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig NR70_Csa_0-3825 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig NR24_Csa_0-1227 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig NR14_Csa_0-1383 was left with a single interval that was filtered out.
12:45:50.630 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R42_0-945 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R403_0-900 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R50_0-900 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R14_0-960 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R10_0-924 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R9_0-936 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R13_0-912 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R20_0-930 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R7_0-957 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R8_0-957 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R410_0-927 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig 3_Mfa_TAS2R413_0-930 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R418P_0-964 was left with a single interval that was filtered out.
12:45:50.631 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R412_0-993 was left with a single interval that was filtered out.
12:45:50.632 WARN FilterIntervals - After applying provided filters, contig Csa_TAS2R415_0-930 was left with a single interval that was filtered out.
12:45:50.632 WARN FilterIntervals - After applying provided filters, contig Mmu_TAS2R414_0-929 was left with a single interval that was filtered out.
12:45:50.632 INFO FilterIntervals - Shutting down engine
[May 24, 2022 12:45:50 PM JST] org.broadinstitute.hellbender.tools.copynumber.FilterIntervals done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=2089287680
***********************************************************************
A USER ERROR has occurred: Bad input: Filtering removed all intervals. Select less strict filtering criteria.
***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.
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Hi 侯旻,
Are you trying to follow this gCNV pipeline? https://gatk.broadinstitute.org/hc/en-us/articles/360035531152--How-to-Call-common-and-rare-germline-copy-number-variants
If so, I noticed that you want to select --padding 0 not --bin-length 0 for PreprocessIntervals.
Let me know if this answers your question.
Best,
Genevieve
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Hi Genevieve Brandt
Thank you for your comment. Yes, I followed by the gCNV pipeline and I selected both bin length and padding as 0. Actually, I tried bin length 860 which is the shortest length of all genes and the program worked well at that time. But the result seems to be too strict when I compared the ploidy results to depth result. For example, one gene showed depth is almost four or five times higher than average sequencing depth while the ploidy result showed 3n for that gene which we expected 4n or more for that gene.
Best regards
Min
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Thank you Min for the update. I will follow up with my team to see if there are any parameters you can use to make the gCNV pipeline more sensitive.
Is this WGS or exome data? Could you also supply all the gCNV commands you ran to get the results that were too strict?
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Hi Genevieve
Thank you so much for the support. I used exome data for the analysis. I attached commands of one species as an example. Because my study species doesn't have WGS so I ran the program in cohort mode (use the same bam file to run AnnotateIntervals). The intervals bed file included taste receptor genes (around 40) and about 83 neutral regions (including 3 regions are from X chromosome: NR83-85) which are supposed to be single copy.
Looking forward to your reply
Best regards
Min
/usr/local/bin/gatk-4.2.5.0/gatk PreprocessIntervals \
-R Sent_3mapping_final.fasta \
-L Sent_TAS2R.bed \
--bin-length 870 \
--padding 0 \
--interval-merging-rule OVERLAPPING_ONLY \
-O pre_Sent.interval_listjava -jar /usr/local/bin/picard.jar AddOrReplaceReadGroups INPUT=Sent_H2_rmd_bqsr.bam OUTPUT=Sent_addRG_1.bam RGID=Sent RGLB=Library1 RGPL=illumina RGPU=Sent RGSM=sample1 VALIDATION_STRINGENCY=LENIENT
java -jar /usr/local/bin/picard.jar AddOrReplaceReadGroups INPUT=Sent_H2_rmd_bqsr.bam OUTPUT=Sent_addRG_2.bam RGID=Sent RGLB=Library2 RGPL=illumina RGPU=Sent RGSM=sample2 VALIDATION_STRINGENCY=LENIENT
samtools index Sent_addRG_1.bam
for i in `ls *bam`; do
sp=`basename ${i} _addRG_1.bam`
/usr/local/bin/gatk-4.2.5.0/gatk CollectReadCounts \
-I ${sp}_addRG_1.bam \
-L pre_${sp}.interval_list \
--interval-merging-rule OVERLAPPING_ONLY \
-format TSV -O ${sp}_addRG1.tsv
donefor i in `ls *bam`; do
sp=`basename ${i} _addRG_1.bam`
/usr/local/bin/gatk-4.2.5.0/gatk AnnotateIntervals -R ${sp}_3mapping_final.fasta -L pre_${sp}.interval_list --interval-merging-rule OVERLAPPING_ONLY -O ann_${sp}1.tsv
donefor i in `ls *bam`; do
sp=`basename ${i} _addRG_1.bam`
/usr/local/bin/gatk-4.2.5.0/gatk FilterIntervals -L pre_${sp}.interval_list --annotated-intervals ann_${sp}1.tsv --interval-merging-rule OVERLAPPING_ONLY -O ${sp}_filter.interval_list
donesamtools index Sent_addRG_2.bam
for i in `ls *bam`; do
sp=`basename ${i} _addRG_2.bam`
/usr/local/bin/gatk-4.2.5.0/gatk CollectReadCounts \
-I ${sp}_addRG_2.bam \
-L pre_${sp}.interval_list \
--interval-merging-rule OVERLAPPING_ONLY \
-format TSV -O ${sp}_addRG2.tsv
donesource activate gatk
/usr/local/bin/gatk-4.2.5.0/gatk DetermineGermlineContigPloidy --input Sent_addRG1.tsv --input Sent_addRG2.tsv --contig-ploidy-priors ploidy_prior.tsv --output Sent_contig --output-prefix Sent
for i in `ls *1.tsv`; do
sp=`basename ${i} _addRG1.tsv`
/usr/local/bin/gatk-4.2.5.0/gatk GermlineCNVCaller --run-mode COHORT -L ${sp}_filter.interval_list --interval-merging-rule OVERLAPPING_ONLY --contig-ploidy-calls ${sp}_contig/${sp}-calls -I ${sp}_addRG1.tsv -I ${sp}_addRG2.tsv --output gcnv_${sp} --output-prefix ${sp}
donefor i in `ls *1.tsv`; do
sp=`basename ${i} _addRG1.tsv`
/usr/local/bin/gatk-4.2.5.0/gatk PostprocessGermlineCNVCalls --calls-shard-path gcnv_${sp}/${sp}-calls --model-shard-path gcnv_${sp}/${sp}-model --sample-index 0 --autosomal-ref-copy-number 2 --allosomal-contig NR83_Can_0-1578 --allosomal-contig NR84_Can_0-2157 --allosomal-contig NR85_Can_0-2154 --output-genotyped-intervals gen_${sp}.vcf --output-genotyped-segments seg_${sp}.vcf --output-denoised-copy-ratios ${sp}_copy.tsv --contig-ploidy-calls ${sp}_contig/${sp}-calls
donevcftools --vcf gen_Sent.vcf --extract-FORMAT-info CN --out Sent_CN
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Thank you Min! I expect that I will be able to get back to you with a follow up Monday or Tuesday next week.
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Thank you for being so supportive!
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Hi Min,
I had the developers take a look at your request to see what they would recommend. They recommended to stick with the default value for --bin-length to avoid your original issue of filtering out all of the intervals.
For your unexpected results, I first wanted to point out that ploidy is calculated on a contig level while copy number is calculated on the gene level. So make sure you are looking at the correct metric for the gene of interest. You may be running into an issue with the quality covering that gene which is why the copy number is lower than expected.
You can check the segments file to see if the quality is high or low. We normally don't see issues with sensitivity when the quality is high. Depending on the event type, here are some example QS thresholds. They are human derived but should give you some basis to go off of:
private int hetDelQSThreshold = 100;
private int homDelQSThreshold = 400;
private int dupeQSThreshold = 50;Any calls with QS < 50 are dubious.
Let us know what you find and if this helps!
Best,
Genevieve
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Hi, Genevieve
Sorry for the late reply and I really appreciate everything you and your team done for me. Thank you so much and thank for the developers' recommendation.
About unexpected results, sorry for the misdiscribed issue. I wanted to know the copy number of each exome data. I also checked the segment vcf files to confirm the QS value and I found that all of them are higher than 55 which means the quality is high.
This week I also discussed with my lab member who is using the GATK to analyze the human sequence data. We thought since I used exome gene and neutral reference region to determine the germline contig ploidy which is supposed to use the contig data or chromosome data (because I don't have the information for any of them), it may be the limitation for the rest analysis steps. What's more, many neutral reference regions are 100 even 1000 times longer than exome regions and according to the bin length I set, those long-length neutral regions will be separated into many parts. Low mapping quality of some parts will influence the average depth when DetermineGermlineContigPloidy is applied. So we decided to filter those low mapping quality reads first maybe by using the MappingQualityReadFilter.
Best regards
Min
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Thanks Min! Keep in mind though that the quality threshold differs for different events. For some events, 55 is not high.
Let us know if you have any other questions.
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Thanks Genevieve and your team. You have been so helpful and supportive. I will keep in mind about the quailty thing.
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You're welcome!
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