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Hello Everyone.
I am sequencing viral genomes by PCR-amplifying them, and then sequencing them on the Illumina platform with 150 x 2 coverage. I generally get about 50,000 reads for each sample using this method.
My amplicon varies in size: this is a retrovirus and is highly mutated, often containing large deletions. The maximum amplicon size is 8.9 kb (unless an insertion has occurred, which could increase the size). I have a ballpark idea of the expected amplicon size, as I screen by gel electrophoresis prior to sequencing. Regardless, the goal is to generate a workflow that accurately assembles the reads into the correct contig size.
For these reasons, I am trying to assemble them using a de novo method. I have tried creating a workflow with Geneious, but keep running into problems. I can't get the get the expected amplicon to assemble into a single contig file. Sometimes it is broken into many different contig files, sometimes pieces are just missing.
I'm working on a desktop mac with 32GB of processing power, so these assemblies take a lot of time, making troubleshooting and playing with parameters a grueling process.
Is anyone familiar with Geneious, or used another program for a similar application. We don't have a bioinformatician in my lab, and I have very limited experience in coding, so user-friendly programs would be ideal. Any help would be greatly appreciated.
Thanks!
I am sequencing viral genomes by PCR-amplifying them, and then sequencing them on the Illumina platform with 150 x 2 coverage. I generally get about 50,000 reads for each sample using this method.
My amplicon varies in size: this is a retrovirus and is highly mutated, often containing large deletions. The maximum amplicon size is 8.9 kb (unless an insertion has occurred, which could increase the size). I have a ballpark idea of the expected amplicon size, as I screen by gel electrophoresis prior to sequencing. Regardless, the goal is to generate a workflow that accurately assembles the reads into the correct contig size.
For these reasons, I am trying to assemble them using a de novo method. I have tried creating a workflow with Geneious, but keep running into problems. I can't get the get the expected amplicon to assemble into a single contig file. Sometimes it is broken into many different contig files, sometimes pieces are just missing.
I'm working on a desktop mac with 32GB of processing power, so these assemblies take a lot of time, making troubleshooting and playing with parameters a grueling process.
Is anyone familiar with Geneious, or used another program for a similar application. We don't have a bioinformatician in my lab, and I have very limited experience in coding, so user-friendly programs would be ideal. Any help would be greatly appreciated.
Thanks!
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