I want to see if there is any GC coverage bias in some paired-end Illumina human exome data. Update This is for a variant calling project. I used the
CollectMultipleMetrics from GATK4 toolkit with
-PROGRAM CollectGcBiasMetrics. An example of the command I used is:
gatk CollectMultipleMetrics -I input.bam -O prefix_dir_name -R input.ref_genome -PROGRAM CollectGcBiasMetrics -PROGRAM CollectAlignmentSummaryMetrics -PROGRAM CollectInsertSizeMetrics -PROGRAM QualityScoreDistribution -PROGRAM MeanQualityByCycle -PROGRAM CollectBaseDistributionByCycle
Update This is my result for the QC coverage bias with one sample highlighted: There seems to be a large bias. I would expect the peak to be around 50% for human. And those weird peaks around 90% are also puzzling to me. On the other hand, I am working with exome data so it might be logical to have a bias towards the higher values of GC% because you have reads on GC-rich exons?
Update first part of useful file (gc_bias.detail_metrics) which shows the reads per bin, as suggested by @Maximilian Press. This I will plot.
## htsjdk.samtools.metrics.StringHeader # CollectMultipleMetrics --INPUT WESP_run/results/aligned/RM8398_MF_S8_MD.sorted.bam --OUTPUT WESP_run/results/aligned/post_alignment_QC/multiple_metrics/RM8398_MF_S8 --PROGRAM CollectAlignmentSummaryMetrics --P ROGRAM CollectBaseDistributionByCycle --PROGRAM CollectInsertSizeMetrics --PROGRAM MeanQualityByCycle --PROGRAM QualityScoreDistribution --PROGRAM CollectGcBiasMetrics --REFERENCE_SEQUENCE /mnt/scratch_dir/deute koe/projects/HP/WESPipe/workflow/resources/reference_genomes/hg38/alt_plus/GCA_000001405.15_GRCh38_full_plus_hs38d1_analysis_set.fna --ASSUME_SORTED true --STOP_AFTER 0 --METRIC_ACCUMULATION_LEVEL ALL_READS --IN CLUDE_UNPAIRED false --VERBOSITY INFO --QUIET false --VALIDATION_STRINGENCY STRICT --COMPRESSION_LEVEL 2 --MAX_RECORDS_IN_RAM 500000 --CREATE_INDEX false --CREATE_MD5_FILE false --GA4GH_CLIENT_SECRETS client_sec rets.json --help false --version false --showHidden false --USE_JDK_DEFLATER false --USE_JDK_INFLATER false ## htsjdk.samtools.metrics.StringHeader # Started on: Sat Dec 03 14:25:15 CET 2022 ## METRICS CLASS picard.analysis.GcBiasDetailMetrics ACCUMULATION_LEVEL READS_USED GC WINDOWS READ_STARTS MEAN_BASE_QUALITY NORMALIZED_COVERAGE ERROR_BAR_WIDTH SAMPLE LIBRARY READ_GROUP All Reads ALL 0 139942 1326 19 0.413544 0.011357 All Reads ALL 1 102412 61 17 0.025996 0.003328 All Reads ALL 2 118957 74 16 0.02715 0.003156 All Reads ALL 3 152181 929 20 0.266429 0.008741 All Reads ALL 4 167732 248 16 0.06453 0.004098 All Reads ALL 5 173319 232 16 0.058421 0.003836 All Reads ALL 6 196365 304 15 0.067567 0.003875 All Reads ALL 7 211217 343 17 0.070875 0.003827 All Reads ALL 8 241125 441 18 0.079822 0.003801 All Reads ALL 9 270272 1068 18 0.172463 0.005277
I have read a paper that uses the CollectGcBiasMetrics on exome data and they magically produce a plot without bias. I say magically because they don't explain how they use CollectGcBiasMetrics.
Update Am I missing something? Should I, for instance, use my target regions in instead of the reference genome when using
CollectGcBiasMetrics? I will try it, but I am not sure this is the correct thing to do... It is hard to find any workflow for exome data with gatk and particularly
CollectGcBiasMetrics, which specifies to use the reference genome.
From the comment @Maximilian Press
Do you have on-instrument diagnostics? Did you run FastQC on the raw reads? What do the other picard metrics look like? Did you manually inspect your alignments (strongly recommended)? Do you actually have coverage of the exome?
I will see if I can get some instrument diagnostics. I did look at the alignments, and as far as I can tell I don't see anything weird? Just that it is very deeply sequenced. I do see some intronic regions, with some reads, but only a few reads compared to the many on the targets, which I think is a consequence of the (too) deep sequencing. The fastqc of the raw reads didn't look troubling, and it did not give me any warnings. The picard metrics I thought did not look out of the ordinary. There is good target coverage. Yes, it is sequenced quite deep. Also, the lab tech did tell me they over-cycled because they wanted to be sure to have all the regions/targets.
Update So this bias might be due to too deep sequencing? And another important question, can I still reliably use this data? If so, I guess I can perform some corrections like suggested here
Any help/suggestions would be very welcome. Thank you in advance.