Now I have a problem and a general question.įor two files I got this error message after the first mpileup commad: The QS annotation not present at gi|33590464|gb|AY244516. We would like to mask these in the consensus sequence as. So far the only thing that seemed to give the LEAST errors was: samtools mpileup -vf refgene.fa > bcftools consensus > consensus. First we will create a bed file containing the locations of low depth regions. Overview As we have seen, the SAMTools suite allows you to manipulate the SAM/BAM files produced by most aligners. Right now I just want to make a consensus sequence but I am at a loss at how to write my bcftools command. Then, I wanted to extract the consensus sequence from the bam file: samtools mpileup -uf reference.fasta my.bam | bcftools call -mv -Oz -o >Ĭat reference.fasta | bcftools consensus > con.fastaĪnd here I am. We will now create a consensus sequence for all isolates by substituting in the alternate alleles into the reference at their respective positions. The results of this command / sequence legth * 100 to have the % genome covered. I mapped some contigs on a Plant reference genome with BWA, sorted the file with samtools and got the coverage by typing: samtools depth my.bam > qry-depth> If you prefer a FASTA format instead of FASTQ, you can use tools like seqtk or fastq_to_fasta to convert the FASTQ file to FASTA format if needed.It's probably the same question over and over but I can't find the solutions. This is a good way to remove low quality reads, or make a BAM file restricted to a single chromosome. Please make sure to replace reference.fasta with the filename of your reference genome and sorted_aligned_reads.bam with the appropriate name of your sorted and indexed BAM file.Īfter running this script, you should obtain the consensus sequence in the consensus.fastq file. bam files - they can be converted into a non-binary format ( SAM format specification here) and can also be ordered and sorted based on the quality of the alignment. vcf2fq: Converts the consensus genotype in VCF format to FASTQ format, representing the consensus sequence.Ĭonsensus.fastq: The output file containing the consensus sequence in FASTQ format. I used the following commands: bwa mem ref.fasta SRR1.fastq SRR2.fastq > bwa.sam samtools view -b -F 4 bwa.sam > bwaaligned.bam samtools index bwaaligned.bam. bam files - they can be converted into a non-binary format ( SAM format specification here) and can also be ordered and sorted based on the quality of the alignment. The results of this command / sequence legth 100 to have the genome covered. The primer is also designed to be self-contained and hands-on, meaning that you only need to install SAMtools, and no other tools, and sample data sets are provided. I am trying to generate consensus sequence from a bam file obtained after mapping SRA reads to a reference genome. samtools depth my.bam > qry-depth> wc -l qry-depth. It is a common practice in genomic studies to use a single reference for mapping, usually the ‘reference genome’ of a speciesa high-quality assembly. If you have run the medakaconsensus pipeline you will have. Sorted_aligned_reads.bam: The sorted and indexed BAM file.īcftools call: Calls the consensus genotype for each position based on the pileup. This primer provides an introduction to SAMtools, and is geared towards those new to next-generation sequence analysis. Author summary Mapping consists in the alignment of reads (i.e., DNA fragments) obtained through high-throughput genome sequencing to a previously assembled reference sequence. As input the core medaka algorithm accepts sequencing reads aligned to an assembly sequence. Wiki Security Insights New issue samtools consensus: Unexpected behaviour in -d option 1700 Closed TCLamnidis opened this issue on 3 comments TCLamnidis commented on edited Linux 5.4.0-64-generic x8664 gcc (Ubuntu 9.4.0-1ubuntu120.04.1) 9.4. This command identifies target regions by examining the continuity of read depth, computes haploid consensus sequences of targets and outputs a SAM with each sequence corresponding to a target. Everything seems fine after we figured out how to filter the variants and get phased sequences by bcftools consensus. f reference.fasta: Specifies the reference genome in FASTA format. samtools targetcut -Q minBaseQ -i inPenalty -0 em0 -1 em1 -2 em2 -f ref in.bam. Samtools mpileup: Generates a pileup of aligned reads at each position in the reference genome. Samtools mpileup -uf reference.fasta sorted_aligned_reads.bam | bcftools call -c | vcf2fq > consensus.fastq
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