## Documentation error in RNAseq workflow

### Posted by Geraldine_VdAuwera on 11 Jun 2014 (4)

We discovered today that we made an error in the documentation article that describes the RNAseq Best Practices workflow. The error is not critical but is likely to cause an increased rate of False Positive calls in your dataset.

The error was made in the description of the "Split & Trim" pre-processing step. We originally wrote that you need to reassign mapping qualities to 60 using the ReassignMappingQuality read filter. However, this causes all MAPQs in the file to be reassigned to 60, whereas what you want to do is reassign MAPQs only for good alignments which STAR identifies with MAPQ 255. This is done with a different read filter, called ReassignOneMappingQuality. The correct command is therefore:

java -jar GenomeAnalysisTK.jar -T SplitNCigarReads -R ref.fasta -I dedupped.bam -o split.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS

In our hands we see a bump in the rate of FP calls from 4% to 8% when the wrong filter is used. We don't see any significant amount of false negatives (lost true positives) with the bad command, although we do see a few more true positives show up in the results of the bad command. So basically the effect is to excessively increase sensitivity, at the expense of specificity, because poorly mapped reads are taken into account with a "good" mapping quality, where they would normally be discarded.

This effect will be stronger in datasets with lower overall quality, so your results may vary. Let us know if you observe any really dramatic effects, but we don't expect that to happen.

To be clear, we do recommend re-processing your data if you can, but if that is not an option, keep in mind how this affects the rate of false positive discovery in your data.

We apologize for this error (which has now been corrected in the documentation) and for the inconvenience it may cause you.

#### sirian

Thanks for the correction! I was actually wondering a little bit why you changed every score.

#### sboyle

Thanks for the correction Geraldine!

#### Geraldine_VdAuwera

@kam This may be a good recommendation to make to the authors of the mappers.

#### GATK Dev Team

###### @gatk_dev

@thatdnaguy Note: future releases may switch to mem at some point. For our prod pipelines this is tied to shift to cloud.
###### 26 Aug 16
@thatdnaguy @exac_exomes Same as first iirc, bwa aln as noted in supplemental doc https://t.co/ziwLjp7iIw
###### 26 Aug 16
@MattBashton Fixed, thanks for reporting!
###### 22 Aug 16
@martenson @pnatarajanmd @openscience @broadinstitute @dgmacarthur Baby steps.
###### 18 Aug 16
@martenson @pnatarajanmd @openscience @broadinstitute @dgmacarthur FWIW tracking feature is gone https://t.co/BsGNmBxQ0W

###### Our favorite tweets from others

it's the nightly build owl for GATK :D https://t.co/OwTRrk6KHA https://t.co/rfmAbdIIQp
###### 11 Aug 16
We're going to make an hg38 version of ExAC. And we'll make @dgmacarthur pay for it. #BioinformaticsCampaignPromises
###### 2 Aug 16
You’re right @gatk_dev honesty is key! About variants manual filtering: “In any case you're probably in for a world of pain.” Ha now I know!
###### 11 Jul 16
.@gatk_dev I like the new documentation index page, the subheading has made my day! :D #doge #GeekHumourFTW https://t.co/9RXnDTMoBm
###### 8 Jul 16
There is no NGS, NG is today so should only be called high-throughput sequencing #CSC #GATKworkshop https://t.co/paHcNimD7o
###### 16 Jun 16
See more of our favorite tweets...