WGS Variant Calling: Variant Filtering and Annotation - Part 2 | Detailed NGS Analysis Workflow

This is the detailed workflow tutorial on how to filter variants and annotate them using GATK's Funcotator tool. In this video I discuss the need of filtering and annotating variants, the questions we may ask once we call variants, two approaches to filter variants i.e. Variant Quality Score Recalibration (VQSR) & hard filtering, the data sources available, genotype refinement and finally talking about Funcotator. Further, I demonstrate how to filter variants at the site level as well apply genotype filters and annotating variants using Funcotator. Lastly, I demonstrate how to wrangle the output from Funcotator and get the annotations in a tabular format which is helpful for further processing and visualization. I hope you find this video helpful! Leave your thoughts in the comment section below!

▸ Code:

▸ Part 1 of this video:

▸ VCF file format video:

▸ GATK hard-filters recommendations for germline variants:

Chapters:
0:00 Intro
0:43 Overview of variant calling steps
1:34 Questions we may want to ask after calling variants
3:00 Variant data sources
5:16 Two approaches to filter variants
8:38 Hard filtering: Site-level filtering
9:14 Hard filtering: Sample-level filtering
10:15 Genotype Refinement
10:54 Data used for today’s demo
11:22 Pre-requisites
12:22 Filtering SNPs
16:54 Filtering INDELS
17:35 Understanding output after filtering (FILTER column)
19:19 Select variants that passed filters
20:39 Exclude variants that failed genotype filters
25:49 GATK Funcotator tool
28:10 Funcotator data sources
29:59 Annotate variants using Functotator
32:07 Understanding output after annotation (Funcotator output)
33:45 Getting annotations into a tabular format

You can show your support and encouragement by buying me a coffee:

To get in touch:

#bioinformagician #bioinformatics #funcotator #variantcalling #variants #gatk #vcf #gvcf #haplotype #alleles #geneticvariants #mutations #gff3 #gff #gtf #sam #bam #phred #fasta #fastq #singlecell #10X #ensembl #biomart #annotationdbi #annotables #affymetrix #microarray #affy #ncbi #genomics #beginners #tutorial #howto #omics #research #biology #GEO #rnaseq #ngs

[julinha_]

I usually never comment on anything, really, but I would be remiss if I didn't mention what a great job you are doing. There are not a lot of videos, or at least none I could really find, that make video tutorials on such specific topics like this. Really good job :) Thank you!

[eyadelbahtety]

Both videos part 1 and 2 are incredible, thank you so much for your dedication and hard work putting this together in such good details. You have helped me significantly understand how to utilise this workflow to process bam files all the way into my variants of interest. Thank you!

[belizg]

Thank you very much for Part 2 and especially for focusing on hard filtering as we requested you to do the video

[chung]

Fantastic! Your presentation is clear and detail, helps me a lot in understanding the hard filtering process😊

[hayatdeen]

i wish i found you sooner, i had to painstakingly browse though tones of GATK web pages to get me started with the Best practice guidelines. you made it clearer

[harshasatuluri]

Thank you ma'am for sharing ur knowledge.... Happy to be ur student form online😊

[mentarikasih]

your tutorial really help me a lot to understand the workflow. If you have experience analyze gwas and also want to make the tutorial, I would appreciate it. Thank you so much ma'am ☺️

[osmandoluca]

@22:00 Instead of doing all that, you can add '-select-genotype "GQ > 10 && DP > 10" \' to the SelectVariants commandline.

[pauladisanto]

You are great! Thanks for this amazing tutorial!

[venkatakrishnakandalai]

Thank you for this very informational video!

[madhavanjn]

Thanks for ur amazing explanation. Plz keep posting more videos, can u make a video on how to use restapi, github, json, aws etc.

[peipei]

Thanks so much for your help this is tremendously helpful! I was wondering where you found the GATK guidelines for the hard filters that you applied? Or if you know them based from experience? I can't seem to find the recommended thresholds anywhere on the GATK website (with the exception of a page that uses QUAL as a filter instead of QD and seems to be outdated). TIA!

[RuqaiyaTasneem-zw]

how to set those filters ? what do all do we have to consider ?

[OmPrakash-obcz]

Any script explanation for diplotype calling?, In the same way as you explained variant calling in a very clear way.

[赵彬彬-is]

Thanks very much for the video. Could you continue to explain Call somatic mutations with Mutect2?

[hadieidgah]

Fantastic work, thank you and it is always worth to watch your videos till the last second. Can one use this pipeline for WES as well?

[alialjizani]

Thank you that was helpful, but what if I have the WES data in form of Excel sheet how can I filter my variants? and is there a tool or a program can do that?

[sgrtools]

This is a great tutorial !.
Can you please make a video on "Call somatic mutations using GATK4 Mutect2"?? and a video on "maftools" - R package to analyze somatic .vcf files??"
Highly appreciated !

[sinugeorge]

Could you please make a video for GTAK-mutect2 variant calling?

[shobhitashah]

Can you help me with annotation of somatic variants? Are tools for germline and somatic annotation same?