# Tagged with #read filters 1 documentation article | 0 announcements | 7 forum discussions

Created 2012-08-11 05:16:06 | Updated 2015-12-19 10:53:18 | Tags: developer downsampling

#### Downsampling is a process by which read depth is reduced, either at a particular position or within a region.

Normal sequencing and alignment protocols can often yield pileups with vast numbers of reads aligned to a single section of the genome in otherwise well-behaved datasets. Because of the frequency of these 'speed bumps', the GATK now downsamples pileup data unless explicitly overridden.

Note that there is also a proportional "downsample to fraction" mechanism that is mostly intended for testing the effect of different overall coverage means on analysis results.

See below for details of how this is implemented and controlled in GATK.

## 1. Downsampling to a target coverage

### Defaults

The GATK's default downsampler (invoked by -dcov) exhibits the following properties:

• The downsampler treats data from each sample independently, so that high coverage in one sample won't negatively impact calling in other samples.
• The downsampler attempts to downsample uniformly across the range spanned by the reads in the pileup.
• The downsampler's memory consumption is proportional to the sampled coverage depth rather than the full coverage depth.

By default, the downsampler is limited to 1000 reads per sample. This value can be adjusted either per-walker or per-run.

### Customizing

From the command line:

• To disable the downsampler, specify -dt NONE.
• To change the default coverage per-sample, specify the desired coverage to the -dcov option.

To modify the walker's default behavior:

• Add the @Downsample interface to the top of your walker. Override the downsampling type by changing the by=<value>. Override the downsampling depth by changing the toCoverage=<value>.

### Algorithm details

The downsampler algorithm is designed to maintain uniform coverage while preserving a low memory footprint in regions of especially deep data. Given an already established pileup, a single-base locus, and a pile of reads with an alignment start of single-base locus + 1, the outline of the algorithm is as follows:

For each sample:

• Select reads with the next alignment start.
• While the number of existing reads + the number of incoming reads is greater than the target sample size:

Now walk backward through each set of reads having the same alignment start. If the count of reads having the same alignment start is > 1, throw out one randomly selected read.

• If we have n slots available where n is >= 1, randomly select n of the incoming reads and add them to the pileup.
• Otherwise, we have zero slots available. Choose the read from the existing pileup with the least alignment start. Throw it out and add one randomly selected read from the new pileup.

## 2. Downsampling to a fraction of the coverage

Reads will be downsampled so the specified fraction remains; e.g. if you specify -dfrac 0.25, three-quarters of the reads will be removed, and the remaining one quarter will be used in the analysis. This method of downsampling is truly unbiased and random. It is typically used to simulate the effect of generating different amounts of sequence data for a given sample. For example, you can use this in a pilot experiment to evaluate how much target coverage you need to aim for in order to obtain enough coverage in all loci of interest.

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Created 2016-01-23 05:03:33 | Updated | Tags: variantfiltration snp gatk

Hi,

I am doing an analysis on chimp variants, and do not have enough variants to train the model in order to perform VQSR. I am using hard filters at this point, as per the recommendation. I keep reading that the recommended cutoff values are only recommendations and that I should expect to tweak the parameters further. However, how do I do this in an effective manner? I'm just not sure how to approach exploring the parameter space.

Should I try out different values around the recommended cutoffs and, down the line, analyze how many mutations I get/calculate certain values for quality control and check that they agree with what is expected (CpG transition rate, and so on)? Is there a way to inspect the data to see whether it is behaving well, or do I have to wait until down the line to see how things are going? Any insight into this would help tremendously.

Thanks! Alva

Created 2015-08-18 12:42:10 | Updated | Tags: haplotypecaller variantfiltration

Hi I am new to using HC. I am just using the HapMap sample NA12878 to validate my pipeline. I ran the following command on a BAM file that I generated. I noticed that certain SNPs were filtered out most of which I can understand the rationale.

java -Xms454m -Xmx3181m -XX:+UseSerialGC -Djava.io.tmpdir=/home/tx/tmpYdAKAC -jar /usr/local/share/gatk/3.4-46-gbc02625/GenomeAnalysisTK.jar \ -R /usr/local/share/genomes/Hsapiens/GRCh37/seq/GRCh37.fa \ -I /home/INDELanalysis/validationrun/NA12878-sort-11_0_15994663-prep.bam \ --dbsnp /usr/local/share/genomes/Hsapiens/GRCh37/variation/dbsnp_138.vcf.gz \ -L 11:1628982-1651500 \ -T HaplotypeCaller \ -o /home/INDELanalysis/validationrun/NA12878-11_0_15994663-raw.vcf.gz

I ran the same analysis using Samtools and the following SNP was in the final filtered output

Samtools output 11 1651228 rs71454096 C G 117 PASS AC=1;AN=2;BQB=0.61207;BaseQRankSum=-0.458;DB;DP=21;DP4=11,0,5,3;FS=7.225;GC=71.29;HOB=0.5;HRun=3;ICB=1;MQ=56.97;MQ0=0;MQ0F=0;MQB=0.395294;MQRankSum=-1.091;MQSB=0.798516;QD=5.57;RPB=0.902014;ReadPosRankSum=-1.162;SGB=-0.651104;VDB=0.0103095;EFF=NON_SYNONYMOUS_CODING(MODERATE|MISSENSE|gCg/gGg|A53G|237|KRTAP5-5|protein_coding|CODING|ENST00000399676|1|G) GT:DP:PL 0/1:19:150,0,158

However when I run it through HC with the above settings the above variant is filtered out of the analysis. When I ran HC with--emitRefConfidence GVCF I got the following output for this variant:

HC output 11 1651228 rs71454096 C G,<NON_REF> 0 . DB;DP=20;MLEAC=0,0;MLEAF=0.00,0.00;MQ=58.22 GT:AD:DP:GQ:PGT:PID:PL:SB 0/0:10,0,0:10:34:0|1:1651120_G_T:0,34,442,34,442,442:10,0,0,0

I am a little confused why it got a QUAL score of 0. So looking nearby I also noticed that there were some sizeable INDELS

11 1651198 . GAGGCTGTGGGGGCTGTGGCTCCGGCTGTGC G,<NON_REF> 0 . DP=25;MLEAC=0,0;MLEAF=0.00,0.00;MQ=58.38 GT:AD:DP:GQ:PL:SB 0/0:14,0,0:14:37:0,37,585,42,589,594:12,2,0,0 11 1651199 rs71025763 A AGGCTGTGGCTCC,<NON_REF> 0 . DB;DP=18;MLEAC=0,0;MLEAF=0.00,0.00;MQ=57.74 GT:AD:DP:GQ:PL:SB 0/0:6,0,0:6:28:0,28,332,28,332,332:5,1,0,0

However neither of these INDELS were called either. Can anyone shed some light as to why nothing is called in this region? If I look at the site 11 1651228 in the bam file I can see that most of the calls were made on the forward strand so there is likely to be some strand bias here and in actual fact we can see that HC called the genotype 0/0 with only 10 reads contributing to this genotype call. I presume the remaining reads were filtered out. However when I look at the pileup for this position I don't understand why over half the reads were filtered

11 1651228 C CCCCGGGGCCCGGCCCGGCGG BBFIFB<FFIIFFFFFFFBBB

Any advice help would be much appreciated.

Created 2015-07-30 17:47:43 | Updated | Tags: overclippedreadfilter

Hello,

I have 51bp reads and am trying to filter out those where about 20-30 bases have been soft-clipped. The command I have been using is:

java -Xmx8g -jar $GATK_JAR -R$REFERENCE -T PrintReads -rf OverclippedRead --filter_is_too_short_value 40 -o /dev/stdout --disable_bam_indexing -I snippet.bam

Using GATK version v3.4-46, human reference hg19.

The problem I'm encountering is that this doesn't actually filter out any reads. I'm looking at many reads with 27 soft-clipped bases and 24 matches, but those are included in the output. Also the log is unambiguous:

-> 0 reads (0.00% of total) failing OverclippedReadFilter MicroScheduler - 0 reads were filtered out during the traversal out of approximately 2474 total reads (0.00%)

Thanks.

Created 2014-07-31 14:05:07 | Updated | Tags: haplotypecaller best-practices

Hi,

I am wondering how HaplotypeCaller in GATK 3.1 handles multimappers? I thought I read that they are passed over for variant calling but stay in the realigned, recalibrated BAM for 'completions sake', like marking duplicates not removing them but cannot find supporting info on the website or from farther afield,

Presumably it is the NotPrimaryAlignmentFilter but there is no info on that posted yet. I know I can output a BAM from HC with haplotype info in there but can I just get reads used in variant calls? Or should I trim the BAM myself to retain reads I want used? I do this for mark duplicates (removed) but for multimappers I would like to know how you define so I can do the same. The reason is for coverage estimates, using bamtobed or such means I take all realigned, recalibrated which is many more lines including multimappers which skews my results.

Thanks,

Bruce.

Created 2014-05-23 14:22:50 | Updated | Tags: mutect variant-calling radseq

Hi, I would like to analyze a dataset consisting of RADseq (Restriction-site Associated DNA) tags from tumor and normal samples. By nature of the technique, all of the reads start at restriction enzyme cut sites in the genome - therefore the assumptions that mutations will be covered by reads from both directions and staggered with respect to position in the read are violated. Is there a way to override the strand bias and clustered position filters in the MuTect pipeline?

Created 2014-03-03 06:56:01 | Updated | Tags:

We've called variants from our own exome data sets. When compaired with the protocol of 1000 genomes, we found that they just marked duplicates and replicates after local realignment and bqsr rather than removing them before, which is the case of ours. Since we wanna use the CHB and CHS as controls, will the distinct calling strategy affect the final output a lot?

Created 2013-06-27 13:07:18 | Updated | Tags:

Are read filters applied before any down sampling?