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How to find mismatches in an assembly

Edited: I'm using Tablet software to view my sorted BAM file and trying to set a feature to show mismatch threshold at which bases on an alignment coverage track are marked. i. e. if the percent of nucleotide differs from the consensus sequence is greater than that % of reads, it would be marked as a feature.

for this, I need to create a BED or GFF3 file from the sorted BAM file, which has the place of every nucleotide in every contig where the nucleotides of mapped reads has variation greater than the defined percent.

for example, I want to have a bed file with a list of positions where 30% of reads has a nucleotide variation from what is assigned to the assembled contig.

sequencing error mismateches
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written by mary • mary

2 answers

Tablet = god no.

What makes you think they are sequencing errors? How do you tell sequencing errors from BAM files? Can you clarify the example? 40% of mapped reads dont show tha same nucleotides as what?

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written by BioinfGuru BioinfGuru

Reads do not show same nucleotides as what? Same as reference nucleotides at some positions? If for a given position you see 60% of reads have reference nucleotide and 40% have another nucleotide at this position, then it is very likely a heterozygous call if this is a diploid genome. There is a way to remove most of the sequencing errors by using a haplotype caller, that calls haplotypes instead of single variants.

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written by Petr Ponomarenko Petr Ponomarenko

Dear friends. I am working on de novo assembly of a prokaryotic cell. What I mean, is alignment mismatches. 40% of mapped reads don't show the same nucleotides as what has been predicted to be in the resulting genome. Look at the situation in the beginning of this contig for example:contig

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written by mary • mary

Hi mary, Which tool are you using to map raw reads to assembled transcripts?

Thanks, I have seen in your question.

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written by Bioinfonext Bioinfonext

I'm working on genomic DNA, not transcripts and I mapped them with bowtie2, then sort and index by samtools

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written by mary • mary

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