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I used AHA in PacBio's smrtanalysis-2.0.1 to scaffold contigs using long-read PacBio data. I now have the output files "scaffold.gml" and "scaffold.fasta". I now realise I have no idea how to use these files and my contigs to generate one long fasta sequence. Can anyone help me out here?
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Hi schroedj,
The scaffold.fasta should contain the most contiguous sequence that the data and algorithm is able to generate. If it isn't "one long fasta sequence" then you need to look into why you're getting the results you are getting, or if AHA is making any difference. There should be very basic stats on the number of scaffolds created, which you can compare to the number of contigs you supplied. Is this what you are asking about?
We look at:
How large is the genome you are trying to assemble? (AHA is only supported up to 200Mb)
How "good" are the contigs going into the AHA? (number of contigs, contig lengths, %of genome, etc.)
How much PacBio Data are you supplying? (My personal rule-of thumb is to give AHA at least 5x coverage, but 2x coverage is the minimum to get any results at all. That is because the "redundancy" parameter is set to 2 by default. This is the minimum number of reads needed to link two contigs).
The parameters to tune AHA can be found in the smrtpipe reference guide:
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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