Pandaseq assembly

[evepy88]

Hi,

I just started to learn how to use Pandaseq and I have some questions which I would like to seek for help.

1) I don't understand the explaination of the algorithm "-t" in the manual. What does the "score" referred to? How to select what score is needed for the assembly?

2) I have include -u in the command line but the unpaired sequences are not saved in any file after the assembly. What should I do? same goes to -g for the log file.

3) How to save the contigs into fastq file instead of fasta?


My appology for my bad english.

Thank you very much,
eve

[mastal]

In reply to part 3) of your question:

I have no experience with Panda-Seq, but most assemblers produce contigs files in fasta format, not fastq.

[westerman]

1) From the code, -t is defined as: "The minimum probability that a sequence must have to match a primer." The default is 0.6. So unless you are primer-matching then '-t' would not be relevant.

As far as I know probability for primer matching is set the same way as it is for overlap. Which, quoting from the FAQ, is "... where there is a mismatch, it is the probability that one base was sequenced correctly and the other was sequenced incorrectly. If both bases have high [input quality] scores (i.e., are probably correct), the chance of the resulting base is low (i.e., is probably incorrect). For more information, see the paper. Also, remember that the PHRED to probability conversion is not linear, so most scores are relatively high. "

2) I have never used '-u' however I am surprised that is not working. Perhaps you can give your full command line? Perhaps there is a simple error in it.

3) use -F ... note that the quality scores are "conceptually different from the input quality scores" and that you should not use the q-scores for further quality filtering. I had one customer use panda-generated fastq files as input into QIIME with very mixed results.

[evepy88]

Hi,

The command line looks like this:

$pandaseq -f N1_R1.fastq -r N1_R2.fastq -u N1_unpaired.fasta -6 -l 140 -o 50 -g N1_log.txt -N -w N1_assembled.fasta > N1_assemble.fasta


Thanks for helping.
eve