Question: Generating consensus sequence from bam file
0
gravatar for elienesantossilva09
28 days ago by
elienesantossilva090 wrote:

Hello, I am trying to generate consensus bam file sequences. For this I used the commands recommended by the link below.

https://samtools.github.io/bcftools/howtos/consensus-sequence.html`][

I did a test and managed to generate a consensus in fasta format. but when I used the command below (first command) I received a message that my sample was considered diploid because I did not specify the ploidy. in my case it is mitochondrial dna and i need to specify in first command. How do I do that?

1 command

bcftools mpileup -Ou -f NC_012920.1.fasta 1-321.bam | bcftools call -mv -Oz -o 1-321.vcf.gz
bcftools index 1-321.vcf.gz

other sequential commands:

     (base)liene@gar:~/Desktop/doc_liene/vcfs$ 
     (base)bcftools norm -f NC_012920.1.fasta 1-321.vcf.gz -o 1-321N.bcf
     Lines   total/split/realigned/skipped: 38/0/3/0
     (base) liene@gar:~/Desktop/doc_liene/vcfs$ 
     bcftools filter --IndelGap 5 1-321N.bcf -Ob -o 1-321NI.bcf
     (base) liene@gar:~/Desktop/doc_liene/vcfs$ 
     cat NC_012920.1.fasta |bcftools consensus 1-321.vcf.gz > 1-321MTconsensus.fa
      Note: the --sample option not given, applying all records
      The site NC_012920.1:8270 overlaps with another variant, skipping...
      The site NC_012920.1:8272 overlaps with another variant, skipping...
      (base) liene@gar:~/Desktop/doc_liene/vcfs

Another question I have is whether I made all the other commands right. although it generated bcf files, they were not used in the consensus extract command syntax. I would like to understand that. Thanks

sequence assembly • 158 views
ADD COMMENTlink modified 25 days ago • written 28 days ago by elienesantossilva090

Thank you very much. It was of great help.

ADD REPLYlink written 25 days ago by elienesantossilva090

Please use ADD COMMENT for comments. If the answer was helpful, consider to upvote and accept:

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ADD REPLYlink written 25 days ago by ATpoint24k

Did you add this to the original question today?

Another question I have is whether I made all the other commands right. although it generated bcf files, they were not used in the consensus extract command syntax. I would like to understand that.

Adding new questions to original post is not going to get them noticed (we don't have a good way to keep track unless you explicitly add a manual time stamp).

ADD REPLYlink written 25 days ago by genomax73k
1
gravatar for finswimmer
28 days ago by
finswimmer12k
Germany
finswimmer12k wrote:

Hello,

the ploidy can be set explicit for a region and sex using a ploidy file. That is a tab delimited file with the columns CHROM, FROM, TO, SEX, PLOIDY. If no ploidy file is specified with --ploidy-file bcftools assumes this default:

X 1 60000 M 1
X 2699521 154931043 M 1
Y 1 59373566 M 1
Y 1 59373566 F 0
MT 1 16569 M 1
MT 1 16569 F 1
*  * *     M 2
*  * *     F 2

So if your mitochondrial reference sequence is name MT you don't need to do anything. There are more defaults you can set with --ploidy. See the output of bcftools call --ploidy list for more information.

although it generated bcf files, they were not used in the consensus extract command syntax. I would like to understand that.

You normalize and filter your vcf file after the calling step, but you take the original vcf file as an input for the consensus creation. I guess this is not what you want to do. Just take the filtered bcf file if this is what you want to do.

fin swimmer

ADD COMMENTlink written 28 days ago by finswimmer12k
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