Plus, you might want to compare tools/methods and compare. There isn’t a Galaxy Training Network tutorial that covers using these tools in detail, but looking at other workflows variant calling tutorials would probably help. It won’t including any base-level variation your read data may have had. samtools mpileup -uf reference.fasta file.bam bcftools call -c vcf2fq > sample.fq. The “consensus sequence” that used to be generated by older versions of Mpileup were encoded and probably not what you are both wanting as a final result (is NOT a fasta “consensus sequence” result based on the variation in your data – what you might think of as a type of “assembly” result).Īlso, using coordinates of regions in a pileup result (or VCF result, or gtf/bed/interval result) to Extract sequences from the genomic sequence will only result in fasta sequence based on that original reference genomic sequence again. I want to create consensus fasta sequence for long-read sequencing BAM files. The tool NormalizeFasta can be used in most cases to standardize the format of fasta datasets. No matter where you get it, it must be an exact match (genome build/source/version) for what you originally mapped against – plus the fasta should be in a very simple format – meaning, no “>” identifier line description content. If you are not sure where to find the fasta version of a pre-indexed reference genome you mapped against, please write back and we can help. These tools do not have built-in indexes like mapping tools. You will probably need to make use of a custom reference genome/transcriptome/exome fasta dataset. Please give these a try and see if it produces the output you each want – these are flexible tools with many options. o Generate the consensus sequence for one diploid individual: samtools mpileup. These tools will call variants (pileup or VCF), fill in reference bases where they are not represented in your data (a few different ways), and generate new consensus sequences given the 1) original reference sequence the variants were called against and the 2) variation output VCF. Extract/print all or sub alignments in SAM or BAM format. This is the easiest solution.Hi & see the choices in the BFCtools tool suite. If your VCF files are from GATK, then recent versions of GATK4 now have FastaAlternateReferenceMaker, which is simple to run on gVCF/VCF files from GATK4. However, it seems that conseqs.fastq (see command below) is missing consensus sequences. Thus, depending on how your VCF files were produced you may or may not be able to use a consensus building method. Hi, I have been trying to use samtools to create consensus sequences. A detailed history explaining the adoption of different symbols as unspecified alternate alleles is documented in an issue on samtools/hts-specs. bcftools however explicitly does not handle any symbolic alleles except, including. Previous versions of samtools used X and as well although this was not so well documented. Meanwhile, VCF4.3 explicitly uses to indicate the unspecified alternate allele. Everything seems fine after we figured out how to filter the variants and get phased sequences by bcftools consensus. This provides us with a way to represent the possibility of having a non-reference allele, and to indicate our confidence either way.” “The first thing you’ll notice, hopefully, is the symbolic allele listed in every record’s ALT field. Briefly, Minimap2 (version 2.17) was used to align reads to a reference sequence (generated by Canu) and SAMtools (version 1.9) was used to generate.BAM files. The VCF format specification by hts-specs is not necessarily followed, in particular by GATK which uses to indicate symbolic alleles in genomic VCFs. Some of the difficulty appears to be inconsistencies in the VCF files produced by different tools. Indeed, bcftools consensus from bcftools should do the trick perfectly well.Īlas, it is not so. In theory, this should be easy: go along the reference and replace the reference base call with the SNP call instead. Different use cases for it exist, one of which is to build phylogenies. References to Oscar and CBC have to do with our computing cluster (named Oscar) and the shorthand for our center (CBC).īuilding a consensus sequence from a VCF file is apparently asked a lot. If you have an id that is not at the start (and is unique to a single sequence header) you simply use if accessionid in. Note: seqrecord can have different tags, check in which one your identifier is located. Note: this post has also been cross-posted to the Center for Computational Biology of Human Disease at Brown, where I currently work. This will go through your sequence records (fasta file) and for each entry check if there is a match with an id from accessionids file. Building a consensus sequence with vcf files
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