WGS Variant Calling: Variant calling with GATK - Part 1 | Detailed NGS Analysis Workflow

[@fesehaabebe-akele2684]

Excellent work!! What makes this young lady standout is that she started with raw data and showed every step in clear terms. I have seen many "tutorials" and almost all started with munged data without showing what was done and how it was done leaving the beginner at a loss. Highly commendable work!

[@enricoperez-kp8zc]

Oh my god! You are awesome. Tu eres la mejor. You are the best. I have found finally someone who can explain the theory of what needs to be done, then do it on the command line, and then explain the reason why to use the terms. The pace is good but I also agree and I would like to suggest to give a bit of time to see what is written in the Terminal. I learnt so much from you in the past two days for watching your videos on GATK variant calling and analysis than the past two years trying to do it by myself. You make writing a script so easy, with explanations along the way. Please, keep doing these types of tutorials, so we can all benefit from these great tutorials. They make my life easier. My next tutorial with you in on RNA-seq. I already have done STAR, which it will be nice if you can do a tutorial, but if you think the way to go is HISAT2 then I am with you. Any tutorial on HISAT-3N soon on your channel? also it will be nice to explain how to install HISAT-3N step by step. I am still in the limbo with this one.

[@kubracelikbas6606]

It would be very nice if you can also provide somatic variant analysis workflow

[@taniadas3301]

Was waiting for this tutorial since so long 🥺

[@PsycheSnacks657]

Many many thanks!. You are helping a lot of folks out there. 😀

[@jeetnanshi4357]

Hi, great information content in the video, just one suggestion, could you use some pointer or animations at each step so that it become interactive and keep track where you are talking. Thanks

[@tiagominuzzi5499]

Great video! Thank you very much. I'm starting to work on this field, this video helped me a lot.

[@srushtikunsavalikar3307]

You are helping a lot ! Thank you so much 🤗

[@emantoraih7700]

You are the best. Is there possibility to make similar videos for somatic mutations? Thanks.

[@mockondo3011]

Great tutorial, the best I’ve seen. I just had one quick question, what if I don’t have a known variants file to perform base quality recalibration? Should I just continue with the next steps using the deduplicated bam file? Hope you can help me with this, in any case, Thanks for the magnificent overview

[@dongholee2144]

did you figure this out?

[@Sjjeien]

Hy i was an aspiring bioinformatician. But by watching all those tutorials i feels that i would surely struggle to get a job in this competative field even though i learned python and r for this masters. I think its better as we are competing with cs grads than lifescience grads... So due to this uncertainity. Im about to take microbiology / biochemistry ... Not all our dream comes true. For me bioinfo was one such dream

[@kumarparas5700]

Excellent Video Ma'am 👍

[@nikver1102]

This is high quality content! Very helpful and crystal clear! Thanks so much! I was wondering... could we use fastp instead of fastqc ? since i think you can also trim with fastp if needed...is that correct? Again, thank you for this content, it is magical 😀

[@Bioinformagician]

I haven't used fastp before. If it does the same thing as FastQC and allows you to perform trimming as well, then go for it.

[@RaviKumar-wg9zm]

Thank you Bioinformagician ma'am

[@Dalibenamor-j8f]

Thank you for this very instructive tutorial. Do we use exactly the same pipeline (packages and tools) to process WES data? thank you in advance

[@harshasatuluri4540]

Thanks, madam, I am waiting for this tutorial!

[@QueenieTsang-h6g]

Thanks for this very informative tutorial! I am trying to run the bwa index reference step, and it looks like it is stuck on the "[bwa_index] construct SA from BWT and Occ ..." step. How long does this step usually take to run?

[@javiflaja4063]

depends on the computational power you have and the size of the reference genome... For a human reference genome, it usually takes between 30min to 1 hr

[@BilalAhmad-gb7ui]

It would be great if you could please make another part to make the publication ready graphs and stuff like annotations from the raw vcf file.

[@Bioinformagician]

Currently working on it!

[@kavoosmomeni4165]

Very helpfull and informative, thank you!

[@ayeshatariq8094]

Hi, thank you so much. Can you please make a video about how to compare two or more vcf files? e.g I want to extract variants that are different in sample vs group

[@Bioinformagician]

Thanks for the suggestion, will surely plan a video on it.

[@isadoramachadoghilardi3168]

Thank you very much for this wonderful explanation!! I have a question: can I identify CNVs in exome sequencing? I love your videos!

[@RaviKumar-wg9zm]

You are doing a great job ma'am. That was really helpful 😊👍

[@andreslavore3928]

Excellent video!! how do you run GATK for poliploid data which variant calling was made using Freebayes? how do you extract several different haplotypes from this kind of data? Thanks

[@johirislam8174]

nice . SO do you have a complete pipeline of WGS data analysis from beginning to end . I mean from sequencing fastq data to variant calling??

[@ОПривет-ъ2ъ]

Amazing tutorial! Thank so much for your work!

[@kajalpanchal8239]

hey! you are really helping a lot ! Thaaaank you so much!

[@moluscosm]

I have a question, does this work demo needs can be run in a 16gb ram computer??'

[@dorrarjaibi]

Thank u foe the explanation ❤ i have a question , if i m using GATK to detect snps in virus , the.BQSR from GAtK resources bundle i use the one for human or for the virus , cause i didn't find anything on the GATK web site

[@MuhammadUsama-u2s]

this tutorial is very nice. I just have one question that my vcf file does not contains the dbsnp id. So is it normal or i am doing any mistake?

[@ayushsafar6289]

you are really helping a lot ! Thaaaank you so much!

[@AnastasiaPetukhova-o9v]

Hi! Could you please make another video on how to call germline CNV and CV or provide me a good tutorial on this topic?

[@Sumit516]

Can this pipeline be used for variant calling in bacterial genomes? Bacterial genome is haploid as opposed to eukaryotes

[@anikeshbanik345]

How much time does the alignment to reference process takes? I am learning it to research about somatic variants in breast cancer cell line.

[@ryanwelch2831]

How long did the gatk BaseRecalibrator algorithm take to make the table? Thank you!

[@Subhash_mahamkali]

does base Qulity recalibration step is very important? beacuse, I am using this pipeline on WGS of sorghum data set. Now I have called the variants without this step ( filtering is done). I used the same vcf file for BQSR step and for some odd reason.. in my .g.vcf file there are no SNPs at all.....

[@himanisareen205]

do you have any video that informs us about the prerequisites that you mentioned like the linux coding and all?

[@ramachandran8106]

Hi, could you upload GWAS tutorial videos please.......

[@moluscosm]

does anywone else gets failed: Operation timed out. when trying to download the read ... ???

[@ДмитрийХолмс-щ9р]

Thank you dear 😊

[@nourlarifi1689]

thank you again for you videos. Could we apply this workflow on scRNAseq data ?

[@kathy_kath]

Hi. Thanks for the tutorial, it helps me a lot! I have a question, how many GB is the resulting .sam file?

[@stemcell1167]

Hi BQSR is too slow any idea how to speed it up? I am using GATK's latest version

[@Harshraj19988]

Perfect. Good job.

[@rajeshsingh-xv7wy]

Ma'am when are you gonna upload the same stuffs for somatic mutation calling?

[@hassanchoudhary9594]

how can we set ploidy argument in GATk , can you share tge script fot that. thanks

[@lauras7670]

Hi, thanks so much for your video. When I run HaplotypeCaller I get the error "Unable to trim uncertain bases without flow order information" and I cannot find anything on the GATK website. My .bam files are validated... I was wondering if you happen to know how to solve this problem. Thanks!

[@tarkkrloglu2406]

Hİ, thank you for tutorials. I have one question. Is there any difference wes and wgs data analysis? Can I use this workflow in wes data analysis?

[@Bioinformagician]

There are some tweaks you'd have to do for WES data. Check out this thread: gatk.broadinstitute.org/hc/en-us/community/posts/4411453286811-Is-there-different-pipelines-between-WGS-variant-calling-and-WES-variant-calling-

[@miannuman8914]

Hello, you are doing a great job, but putting the command in the terminal do it a little bit slow so we can get it easily. hope you understand my point.

[@sakshimehta5467]

hi, could you please tell how do we remove genome duplication in our sample?

[@leearmstrong1581]

Could you make a video on using the limma package for gpr files from GEO? It would be so helpful. Or do you do private tuition? Thanks a million!

[@taniadas3301]

Even I would like to attend if she conducts tuition

[@Bioinformagician]

I will surely consider making a video using limma package and no, I do not provide private or any type of tutoring. Thanks!

[@pratibhagour4336]

Thanks a ton for putting up this video.. Could you please make videos on bi-sulfite sequencing data analysis and also chip-seq data analysis? Thanks in advance :)

[@Bioinformagician]

Sure, definitely have plans to make videos covering these topics :)

[@silvereyes000]

From where did she get the reference genome? She didn't mention anything about it. Anyone else knows?

[@Grzegorz-f1b]

❤❤❤ Thanks You for This learn 👌💪🙏

[@beatricefulton4581]

This is really helpful thank you! Your videos are amazing. Unfortunately, I am having trouble getting CreateSequenceDictionary to work. saying :Neither file nor parent directory exist

[@nourlarifi1689]

I have the same issue how did u solve it plz ?

[@diegopolancoalonso4507]

me too, did you find a way to solve it?@@nourlarifi1689

[@vinaydeep26]

GATK HaplotypeCaller runs very slow. any tips to make it work fast?

[@kubracelikbas6606]

Thanks so much 🌸

[@desaishailesh3527]

Haplotype caller is taking more time, is there problem, i am using GATK 4.0, its going through each chromosome and position, i have 32 GB system though, its more than 3 hours and still have not finished process of till chromosome 2 of one whole genome

[@NishantShekhar-k5m]

Could you please tell... Is 16 gb ram is enough for running gatk?

[@riyakokate9333]

How to add executable to the path? I am using Windows for running this. Would you please tell me how to use Docker to run this commands?

[@kabongontumba9492]

Amazing amazing

[@UmerBaig117]

At 37:27, you did not use the $ sign with the variable data "{data}/recal_data.table". will this variable work without using the $ sign? Can you tell the difference between using HaplotypeCaller for One sample Nad Using HaplotypeCaller with -ERC gVCF mode. And when to use which one?

[@Bioinformagician]

GATK recommends to run Haplotype caller in GVCF mode when you are running more than one sample. With just one sample, running HaplotypeCaller as normal should be sufficient.

[@Hypertyz]

Thanks a lot :D

[@GüllüElifÖzdemir]

Hello, I am having a serioud problem. I am performed your video content from start to finish. However, when performing fastqc step, I constantly get an error that the middle line didn't start with + for the SRR062634_2_filt.fast.gz file. Then, instead of performing this step on the command line, I did this step manually in the fastqc tool, the proces was completed but this time it gave an error in the base sequence quality of the SRR062634_1 file. So I couldn't get the same fastqc results as you. I continued to perform the steps, but after alignment, I could not get any result in the samtools view and flagstat steps. Could you please help me solve this problem? I would also like to thank you for preparing such a nice content.

[@naveedkhan-fi6ux]

It is great work.... its great to watch..... would you please also make a video for 3000 rice genome project and to explain how we can perform SNP variation/haplotype variation within the 3000 rice lines (not just compare two one line with the reference). I also want to perform haplotype analysis for my target gene using 3000 Rice genome project and want to check the haplotype diversity within 3000 rice lines. Anyone have some idea on it..... i would appreciate if someone can help me out

[@mananchelvan157]

Hi, any updates on doing SNP analysis on 3000 lines?

[@naveedkhan-fi6ux]

@@mananchelvan157 no dear, still no update. Maybe this is not her area of interest. I also really need this analysis

[@ayushsafar6289]

what about SRR __ 2 ?

[@RuqaiyaTasneem-z5w]

your bases at the end are of low quality so why not trim it ?

[@ayushsafar6289]

could you help about ancestry

[@nishapaudel5572]

Getting started with GATK4 Follow. GATK - properly pronounced "Gee-ay-tee-kay" (/dʒi•eɪ•ti•keɪ/) and not "Gat-kay" (/ɡæt•keɪ/) - stands for Genome Analysis Toolkit.

[@MsZhang666]

really really really helpful !!!!! I succeed !!! hhhhh😘

[@wajidiqbalwajidiqbal13]

It would be great if you could please make another part to make the publication ready graphs and stuff like annotations from the raw vcf file

[@Bioinformagician]

Thanks for suggestion!