step wise pipeline for SNP analysis (illumina Ampliseq sequencing)

Question

[rebecca08238]

hello everyone!

I am a beginner in data analysis...

1. Can anyone provide the pipeline for SNP analysis, please? (we used Illumina Ampliseq kit for library preparation) We used human DNA sample.
2. How we can calculate Illumina Ampliseq sequencing coverage?

Thank you.

[gsk1185]

your analysis looks correct. i dont have indepth knowledge regarding this kind of study.

you can see refer this.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262760/pdf/gim201465a.pdf

[rebecca08238]

Thank you so much for your reply! I tried mosdepth but it dint work. I used the following command:

mosdepth -t 2 --by capture.bed $sample-output-prefix sample.exome.bam

capture.bed (here I had given my Ampli designBed file and in exome.bam ( I had given my bam file) but this command is not working.

[gsk1185]

mosdepth --by amplicon.bed --thresholds 1,3,5,8,10,20,30 outputfolder sample_Sorted.bam

will give you coverage at different X.

[rebecca08238]

I used above-mentioned command but still, it's not working.

[gsk1185]

its working for me make sure us ban is sorted & also check bed coordinated are matching with fasta used for mapping eg. in fasta there is chr1 in your bed its represented as 1 the you need to change your bed.

[rebecca08238]

ok, I will try again. Thanks!

[jackyen]

Hello, I came across this and is trying to build a pipeline for illumina's ampliseq kit, too. I saw from another post that "soft clipping" should be done before the variant calling step. I'm wondering if someone could explain a bit more. I've heard BBMap could deal with this a bit better than BWA. Thanks

[GenoMax]

Soft-clipping happens during alignment. Aligners will drop part of the read if it does not align.

[jackyen]

Thanks! Is there's a best practice for Illumina's Ampliseq assay? It seems it's very similar to exome except for the mark duplicate step.

[rebecca08238]

Can you please explain about soft clipping...I dont have any idea about this.

[GenoMax]

See this answer: A: What are soft clipped reads??

Answer 1

[gsk1185]

if you have .bed file & bam file you can calculate using mosdepth. https://github.com/brentp/mosdepth

[rebecca08238]

Thanks for your reply! Can you please suggest me pipeline for SNP analysis? Let me tell you in brief what I did after getting raw data from Illumina Miseq. reference genome used: hg19 1st step: Mapping was done by using BWA mem 2nd step: Conversion of Sam to Bam file (using a fixmate command) 3rd step Bam sorting& Bam indexing 4th step: Variant calling using bcftools (v1.9) we created 2 groups files: group1 Normal 30 Individuals and group 2: syndromic patients total 60 individuals in second group. Is this a correct way to generate vcf files or we have to make vcf files for each sample of a particular group and then combining all samples groupwise? So now we have two vcf files: group1.vcf & group2.vcf, I used SnpEff for annotation of these two groups files. I observed that some of the SNPs are present in the control group and syndromic group too, So should I remove those SNPs which are common in both groups using bedtools subtract command? please let me know that I am going in a proper way or not.

I will highly appreciate your help!