Hi all,
We have sequenced a bacterial genome. It is a denovo sequencing. We need to take the sequence of it and use that as a reference and call snp's from 4 more samples which belonging to same sps. What are the steps to get a draft genome?
I have found tools for calling snp's from draft genomes but can anyone suggest some easier user friendly tools as some tools are difficult to understand and implement.
Thanks in advance.
Ramya
Hi Gadicherya,
As mentioned above ,you will need to use bowtie/Star/BWA or any other similar alignment tool.
1.To build the index of your reference , in this case your assembled contigs/scaffolds
2.Align them using bowtie and create a bam file (http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml)
3. Once you obtain your bam file you can call snps using samtools ()
for eg :samtools mpileup -uD -f Ref.fasta bwa.sorted.bam /data/snp_calling/RAL391_full_bwa.sorted.bam | bcftools view -bvcg - > SNPs_samtools.raw.bcf
-u tells it to output into an uncompressed bcf file (rather than compressed)
-D tells it to keep read depth for each sample
-f tells it that the next argument is going to be the reference genome file
The -b flag tells it to output to BCF format (rather than VCF); -c tells it to do SNP calling, and -v tells it to only output potential variant sites (i.e., exclude monomorphic ones); and -g tells it to call genotypes for each sample in addition to just calling SNPs
bcftools view SNPs_samtools.raw.bcf| vcfutils.pl varFilter -D100
> SNPs_samtools.raw.vcf
This line converts the BCF file into a VCF file (a flat text file rather than a binary, making it a lot easier to view), and then we pipe that into vcfutils.pl with the varFilter -D100 option, which filters out SNPs that had read depth higher than 100
I hope this is can help you in your analysis.
Cheers
Shab
Hi,
I think the above suggestions regarding SNP calling are very good and should do the job for you. But if your reference has to assembled first for SNP calling, then its worthwhile to invest some good time in creating a really good de novo assembly (ie draft genome) from your sequencing reads.
Before you begin, make sure that you have quality filtered your raw data. Then install and use a software that would denovo assemble the quality filtered raw reads. Velvet, SOAPdenovo, Edena, IDBA_UD, Masurca are few popular options (and I have used them personally), but there are numerous others available and new ones being published from time to time. Alternatively, you can use proprietary software like CLC workbench (which I have no experience with).
You can start with Velvet , Edena and IDBA_UD, since they (and others mentioned above) are quite easy to use, but remember that these analyses are computationally expensive. These tools would generate Contigs/Scaffolds, ie, fragments of the genome which couldn't be extended further. The outputs of each program will be different, which is why you should run multiple tools with different parameters to find the best combination that gives you the best assembly, ie, bigger total assembly size and in fewer fragments.
While you are doing the assembly, do read the GAGE and GAGE-B articles, which discuss and benchmark performances of different assemblers.
Once you have a satisfactory assembly (draft genome), you can proceed towards calling variations of other samples with the draft as a reference, following the aforementioned suggestions.
PS: Completely finishing a draft genome to create a single supercontig is beyond bioinformatics, and requires specific experiments, however you can use programs like CISA and ABACAS which can take a related genome and order your contigs, filling the gap with Ns to give it a more complete feel (?), but that would still remain a draft genome.
Thankyou Giuseppe, Shab and Anupam for the information you have given me.. This will be of great help to me.. i shall try out the ways which you have suggested.
Thanks for your time..
Will the SAMtool and Bowtie option support ion torrent single reads for bacterial genomes? also once i get the SNP's how do i annotate them and find out whther they fall into the coding or noncoding region?
Thankyou
Hi, just a point to add.
About the assembly you may wish to try BUSCO ( http://busco.ezlab.org/ ) to get some metrics about the assembly that are beyond N50.
PS: do not mistake bowtie with bowtie2, bowtie2 is much better than bowtie.
there is an alternative to galaxy project server focused on Microbiology
here is the link
https://orione.crs4.it/
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