Greetings! My partners and I got this homework. We're not specialized in bioinformatics or anything (pretty amateurs). And we're struggling with some tools. If there is a good soul who would help us my faith in humanity will be.. wait, I never loose faith in humanity, but we would be very thankful.
Our homework consist on the next..
Our data is paired end reads from mother, father and daughter (R1 and R2 of each), by mapping the 3 sets, compare to a proper reference genome and with the right strategies find sites with strong evidence of polymorphism. Utilize VCF for analyzing SNPs, indel, MNP, complex (multiple internal alternative alleles), and the number of the 5 genes with the most polymorphic sites. Including results only when probabilities of a false positive are 1 on 10000 of the Qual column on VCF
Ok.. we found this link with the steps.. \ the workflow is on the link (https://usegalaxy.org/u/coursera/p/genomic-data-science-with-galaxyidentify-polymorphic-sites)
Step 1, step 2 and step 3 are completed by now. Can someone please give a brief explanation of why each step is made in stage 1? (stage 2 is pretty clear though). And how to utilize our data.
Identify DNA polymorphic sites
*This page includes description about analyses of DNA polymorphic sites of father-mother-child sequencing samples:
step 1: load data - the data are loaded from local files, set "fastqsanger" format and "hg19" database on the starting page
step 2: check quality of all sequencing files - use FastQC tool (version: 0.63) to check quality of the sequencing
step 3: mapping - use BWA-MEM tool (version: 0.1) to map sequence to reference genome (choose hg19 as reference), paired end
step 4: add or replace read groups - label each group (the mapping file) using AddOrReplaceReadGroup (version: 1.126.0)
step 5: merge 3 individual mapping files - use MergeSamFiles (version: 1.126.0)
step 6: filter - using filter tools: Filter (version: 1.126.0, remove low quality mapping), MarkDuplicates (version: 1.126.0, filter out duplicated mapping), CleanSam (version: 1.126.0)
step 7: identify polymorphic sites - using FreeBayes tool (version: 0.4) to identify polymorphic sites base on hg19 genome
step 8: filter out false positive sites - using VCFfilter (version: 0.0.3) to select sites where the chance of a false positive call is 1 in 10,000 or better.
step 9: extract workflow and download final vcf file for further analyses.
Stage 2 - analyze data of polymorphic sites based on vcf file
step 10: load data - set format as "vcf", genomic database as hg19
step 11: identify number of snp, mnp, del, ins or complex - using VCFfilter tool (version:0.0.3 ) to select different types of polymorphism (for example: -f "TYPE = snp", select snp only), then using Filter tool (version: 1.1.0) to find duplicated polymorphisms
step 12: identify genes with polymorphic sites - using ANNOVAR Annotate VCF tool (version: 0.1) to annotate the vcf file in step 10
step 13: count polymorphic sites for each gene - using Group tool (version: 2.1.0, by gene name) to count number of polymorphic sites for each gene
step 14: sort results in step 13 using Sort tool (version: 1.0.3, by descending).* ...................................