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De novo assembly for phylogenetic analysis

Hi all,

I have sequencing data from a set of samples of a specific virus. The end goal is include these samples in a larger phylogenetic analysis of this virus and I would like to capture any diversity among my samples. I would like to perform de novo assembly, but I am a bit unsure about which steps are appropriate. So far I am

  • trimming adapters and low quality reads
    • assembling contigs from my fastq files using megahit
  • aligning these contigs back to a reference genome using bwa and samtools

Below are questions about potential extra steps/clarifications

  • Should I also align my reads back to my assembled contigs?
  • What would be appropriate to pull out as a consensus sequence?
  • Is there a way/should I perform a sort of multiple alignment with all of my samples at once?
  • How is best to get coverage statistics of my reads to my consensus sequence?

I appreciate any guidance anyone has!

phylogenetics genomics
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updated by shelkmike shelkmike • written by isag • isag

metagenomic sequencing data from a set of samples of a specific virus

Can you clarify if the sequence is from a single virus species or a mix of species/organisms. Metagenomic sequencing would generally imply a mix.

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written by GenoMax GenoMax

It is for a single virus species

• 0 views • link
written by isag • isag

2 answers

Yes, align reads back to contigs—this lets you polish assemblies and assess coverage. Pull the longest/best-assembled contig as consensus, or use reference-guided consensus if divergence is low.

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written by Priyanka_Dr.OmicsLabs • Priyanka_Dr.OmicsLabs

Since you used MEGAHIT, I assume your reads are short and highly accurate. If this is true, there is no reason to align them to the assemblies and do polishing. However, if you want to calculate contig coverage, you should align the reads and then use a tool like Mosdepth or Samtools.
To find a contig (or contigs) that correspond to the viral genome, you may align a reference genome to the assemblies by BLAST.
For multiple alignment, you can use, for example, the online version of MAFFT (https://mafft.cbrc.jp/). It has a useful option "Adjust direction according to the first sequence" that automatically rotates sequences that were reverse complement in the assembly.

• 0 views • link
written by shelkmike shelkmike

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