Question

FASTA to SAM for Tablet Assembly Visulisations

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5.3 years ago

peflanag • 0

Hi,

I'm very new to the world of terminal command and bioinformatics. I currently use ONT for WGS in my lab. I have the FASTQ and FASTA files of my sequence reads that have been basecalled and trimmed with albacore and porechop. y query to the community is:

The file containing the FASTA reads I have used with a reference FASTA file to perform nucmer to generate a delta file that I can input into the web app Assemblytics. However I am now wondering if I need to use assembled files first before using Assemblytics.

I am also using unicycler to align the long reads to generate an assembled file. The output of which is a FASTA file. I want to visualise the assembly in Tablet but the input format has to be SAM or indexed BAM. Can I easily convert the assembled FASTA file to one of these formats? If so how?

I am using macOS

Cheers,

Peter

Assembly next-gen alignment sequencing • 1.7k views

ADD COMMENT • link 5.3 years ago by peflanag • 0

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It may be best if you spent some time learning command line basics. After doing that you could use minimap2 to align your data to the reference which will generate alignments in BAM format. You can then visualize them in genome browser of your choice.

ADD REPLY • link 5.3 years ago by GenoMax 141k

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Cheers for the quick reply.

I'm using minimap 2 in the Unicycler command " unicycler -l FASTQ.gz -o OUT_DIR " when generating the aligned file. However the output is in FASTA and the visualiser Tablet wants it in SAM or indexed BAM. Thats why I'm wondering can I convert the aligned outputted FASTA into one of these other formats.

ADD REPLY • link 5.3 years ago by peflanag • 0

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unicycler is an assembly pipeline. If you look in read_alignment/ output folder there should be long_read_alignments.sam file (according to unicycler manual). You can try using that to visualize the data.

ADD REPLY • link 5.3 years ago by GenoMax 141k

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Oh cheers, I didn't see that in my output folder! I appreciate the help.

ADD REPLY • link 5.3 years ago by peflanag • 0

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So are you saying that you don't have that .sam file?

Edit: Looks like you will need to run unicycler with --keep 3 option to generate that file.

ADD REPLY • link 5.3 years ago by GenoMax 141k

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I was never using the --keep command. Which I assumed kept everything but can't seem to find the .sam file. I am re-running the unicycler command now with the --keep 3 command

ADD REPLY • link 5.3 years ago by peflanag • 0

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I tried again with --keep 2 and --keep 3 but it hasn't worked for either. I think there was a typo on the github website. the text said --keep two but the table said --keep 3. I have posted on the github issues page.

Cheers for your help!

ADD REPLY • link 5.3 years ago by peflanag • 0