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Recommended but not required that you first complete the trios tutorial and use the data you generated here. Alternatively, canned data provided. |
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As we discussed during some of our variant calling, the majority of the reads, and bases within reads, will map perfectly with the reference genome (unless you are using a reference genome that needs some improvement in which case, you should work towards that end possibly with the spades or novel DNA tutorials as command line jump offs. As so much of the reads are just telling you what you already know, it is possible to design your experiments to target reads to a given location or sets of locations in the genome via sequence capture. One of the most common things to target is the exome, or a portion of the exome as mutations in exons are more likely to have a functional consequence than mutations in introns or intergenic regions.
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Picard is another tool like like gatk also put out by the broad. As of gatk version 4.0, gatk began bundling picard with all of its distributions. This means if you have already done the gatk tutorial you already have access to picard! Alternatively, as picard is just a subset of the gatk package, it may be of interest for you to only install picard tools rather than the full suite. Further, conda offers a "picard-slim" packaging that includes most of the picard tools, but not those few that would otherwise require an associated R program.
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If you wish to install picard as part of the gatk package (allows you to access commands via gatk toolname)
Code Block language bash title As was done in our gatk tutorial, the commands for putting gatk in its own environment and activating it are below. The gatk tutorial contains slightly more information about this installation if you are interested. conda create --name GVA-gatk -c conda-forge -c bioconda gatk4=4.4.0 conda activate GVA-gatk gatk --version
No Format The Genome Analysis Toolkit (GATK) v4.24.60.10 HTSJDK Version: 23.240.15 Picard Version: 23.270.10
If you wish to install picard as a stand alone package (allows you to access commands via picard toolname). Since the only reason that jumps out at me to do this is to not "clutter" things, we'll go with the "slim" package, though changing to the full picard package would not be difficult.
Code Block language bash conda create --name GVA-picard -c conda-forge -c bioconda picard-slim conda activate GVA-picard picard --version
The version command above will actually print the picard help file (aka list of all picard tools). When conda was downloading the package, it appears we are getting version 23.270.30, but I don't see any way to directly access picard's installed version from the command line, though individual tool's version can be access via
picard ToolName --version.
The listing of all of picard's tools tells us that we have successfully installed picard though.
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To run CollectHsMetrics, there are three prerequisites: 1) A bam file and 2) a list of the genomic intervals that were to be captured and 3) the reference (.fa). As you would guess, the BAM and interval list both have to be based on exactly the same genomic reference file.
For our tutorial, the bam files are one of these:
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12892.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12891.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam |
I've started with one of Illumina's target capture definitions (the vendor of your capture kit will provide this) but since the bam files only represent chr20 data I've created a target definitions file from chr20 only as well. Here they are:
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.chr20.reduced.withhead.intervallist /corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.reduced.withhead.intervallist |
And the relevant reference is:
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa /corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa.fai |
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As mentioned yesterday, you can not copy from the BioITeam (because it is on corral-repl) while on an idev node. Logout of your idev session, copy the files. |
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mkdir $SCRATCH/GVA_Exome_Capture cd $SCRATCH/GVA_Exome_Capture cp $SCRATCH/GVA_Human_trios/raw_files/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam . cp $SCRATCH/GVA_Human_trios/raw_files/target_intervals.chr20.reduced.withhead.intervallist . cp $SCRATCH/GVA_Human_trios/raw_files/ref/hs37d5.fa . cp $SCRATCH/GVA_Human_trios/raw_files/ref/hs37d5.fa.fai . |
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mkdir $SCRATCH/GVA_Exome_Capture cd $SCRATCH/GVA_Exome_Capture cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12892.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12891.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.chr20.reduced.withhead.intervallist . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.reduced.withhead.intervallist . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa . cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa.fai . |
Using Picard's "CollectHsMetrics" function to evaluate capture
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Warning | ||
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Make sure you are on an idev node with hostname or showq -u commands. Additional information for launching an idev node is found here. |
The following command is rather large, note that there are a total of 4 commands in the next window: 2 corresponding to loading the correct environment on the compute node, and 2 corresponding to CollectHsMetrics commands. Make sure your copy paste does not introduce additional line returns.
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conda activate GVA-gatk gatk CollectHsMetrics BAIT_INTERVALS=target_intervals.chr20.reduced.withhead.intervallist TARGET_INTERVALS=target_intervals.chr20.reduced.withhead.intervallist INPUT=NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam REFERENCE_SEQUENCE=hs37d5.fa OUTPUT=exome.picard.stats PER_TARGET_COVERAGE=exome.pertarget.stats # conda activate GVA-picard picard CollectHsMetrics BAIT_INTERVALS=target_intervals.chr20.reduced.withhead.intervallist TARGET_INTERVALS=target_intervals.chr20.reduced.withhead.intervallist INPUT=NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam REFERENCE_SEQUENCE=hs37d5.fa OUTPUT=exome.picard.stats PER_TARGET_COVERAGE=exome.pertarget.stats |
Both commands will produce identical results, and nearly identical output to the screen when run (provided you have installed things correctly). Your output is expected to be similar to this which was observed last year:
No Format |
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INFO 2022-06-22 23:04:42 CollectHsMetrics Processed 1,000,000 records. Elapsed time: 00:00:53s. Time for last 1,000,000: 53s. Last read position: 20:14,922,373 INFO 2022-06-22 23:05:31 CollectHsMetrics Processed 2,000,000 records. Elapsed time: 00:01:43s. Time for last 1,000,000: 49s. Last read position: 20:32,404,938 INFO 2022-06-22 23:06:24 CollectHsMetrics Processed 3,000,000 records. Elapsed time: 00:02:36s. Time for last 1,000,000: 52s. Last read position: 20:43,530,401 INFO 2022-06-22 23:07:16 CollectHsMetrics Processed 4,000,000 records. Elapsed time: 00:03:27s. Time for last 1,000,000: 51s. Last read position: 20:56,062,712 INFO 2022-06-22 23:07:40 TheoreticalSensitivity Creating Roulette Wheel INFO 2022-06-22 23:07:41 TheoreticalSensitivity Calculating quality sums from quality sampler INFO 2022-06-22 23:07:41 TheoreticalSensitivity 0 sampling iterations completed INFO 2022-06-22 23:07:41 TheoreticalSensitivity 1000 sampling iterations completed INFO 2022-06-22 23:07:41 TheoreticalSensitivity 2000 sampling iterations completed INFO 2022-06-22 23:07:42 TheoreticalSensitivity 3000 sampling iterations completed INFO 2022-06-22 23:07:42 TheoreticalSensitivity 4000 sampling iterations completed INFO 2022-06-22 23:07:43 TheoreticalSensitivity 5000 sampling iterations completed INFO 2022-06-22 23:07:43 TheoreticalSensitivity 6000 sampling iterations completed INFO 2022-06-22 23:07:44 TheoreticalSensitivity 7000 sampling iterations completed INFO 2022-06-22 23:07:44 TheoreticalSensitivity 8000 sampling iterations completed INFO 2022-06-22 23:07:45 TheoreticalSensitivity 9000 sampling iterations completed INFO 2022-06-22 23:07:45 TheoreticalSensitivity Calculating theoretical het sensitivity INFO 2022-06-22 23:07:46 TargetMetricsCollector Calculating GC metrics [Wed Jun 22 23:07:47 CDT 2022] picard.analysis.directed.CollectHsMetrics done. Elapsed time: 4.09 minutes. Runtime.totalMemory()=1581252608 Tool returned: 0 |
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Here is the output of the above command for a 2023 run, DO NOT! paste this into the command line.
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BAIT_SET target_intervals
BAIT_TERRITORY 1843371
BAIT_DESIGN_EFFICIENCY 1
ON_BAIT_BASES 94769476
NEAR_BAIT_BASES 54852614
OFF_BAIT_BASES 160815315
PCT_SELECTED_BASES 0.481972
PCT_OFF_BAIT 0.518028
ON_BAIT_VS_SELECTED 0.633392
MEAN_BAIT_COVERAGE 51.410962
PCT_USABLE_BASES_ON_BAIT 0.272266
PCT_USABLE_BASES_ON_TARGET 0.212359
FOLD_ENRICHMENT 519.58801
HS_LIBRARY_SIZE 5325189
HS_PENALTY_10X 225.120249
HS_PENALTY_20X -1
HS_PENALTY_30X -1
HS_PENALTY_40X -1
HS_PENALTY_50X -1
HS_PENALTY_100X -1
TARGET_TERRITORY 1843371
GENOME_SIZE 3137454505
TOTAL_READS 4579959
PF_READS 4579959
PF_BASES 348076884
PF_UNIQUE_READS 4208881
PF_UQ_READS_ALIGNED 4161913
PF_BASES_ALIGNED 310437405
PF_UQ_BASES_ALIGNED 286584646
ON_TARGET_BASES 73917336
PCT_PF_READS 1
PCT_PF_UQ_READS 0.918978
PCT_PF_UQ_READS_ALIGNED 0.988841
MEAN_TARGET_COVERAGE 40.099001
MEDIAN_TARGET_COVERAGE 7
MAX_TARGET_COVERAGE 1293
MIN_TARGET_COVERAGE 0
ZERO_CVG_TARGETS_PCT 0.005348
PCT_EXC_DUPE 0.076836
PCT_EXC_ADAPTER 0
PCT_EXC_MAPQ 0.018709
PCT_EXC_BASEQ 0.04137
PCT_EXC_OVERLAP 0.084969
PCT_EXC_OFF_TARGET 0.540013
FOLD_80_BASE_PENALTY 20.049501
PCT_TARGET_BASES_1X 0.924958
PCT_TARGET_BASES_2X 0.820112
PCT_TARGET_BASES_10X 0.473017
PCT_TARGET_BASES_20X 0.423769
PCT_TARGET_BASES_30X 0.385684
PCT_TARGET_BASES_40X 0.34762
PCT_TARGET_BASES_50X 0.308718
PCT_TARGET_BASES_100X 0.137769
PCT_TARGET_BASES_250X 0.011475
PCT_TARGET_BASES_500X 0.000228
PCT_TARGET_BASES_1000X 0.000022
PCT_TARGET_BASES_2500X 0
PCT_TARGET_BASES_5000X 0
PCT_TARGET_BASES_10000X 0
PCT_TARGET_BASES_25000X 0
PCT_TARGET_BASES_50000X 0
PCT_TARGET_BASES_100000X 0
AT_DROPOUT 2.023144
GC_DROPOUT 10.378868
HET_SNP_SENSITIVITY 0.720608
HET_SNP_Q 6
SAMPLE
LIBRARY
READ_GROUP |
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It is rare that you ever want to work with a single sample. While this format is nice for a single sample, comparing the same data across multiple samples would not be the easiest to do with this format. Instead, putting this information to a file, then using grep and awk you could make a small table of the specific information you want. |
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These results were based on sample NA12878. How do the other 2 samples (NA12891, and NA12892) from the trios tutorial compare for their enrichment? If you are unsure how differences in enrichment could effect analysis, this is a great discussion topic.
Return to GVA2022GVA2023 course page.