Suppose I supply Newbler my own trimming points, so that a new sff file is created, from the original sff file, now with this new information. Additionally I also supply files with adaptor and possible contaminants sequences through the -vs and -vt command options.
In this case will my trimming points appear under the columns "Orig Trimpoints Orig Trimmed Length", and any alterations Newbler does based on the -vs and -vt files I supplied, under the columns "Trimpoints Used Used Trimmed Length"?
Or do the original trimming points, i.e. the ones the 454 sequencing platform originally set by including under-case characters, still prevail and appear under "Orig Trimpoints Orig Trimmed Length", while the ones I set appear under "Trimpoints Used Used Trimmed Length"?
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In this case will my trimming points appear under the columns "Orig Trimpoints Orig Trimmed Length", and any alterations Newbler does based on the -vs and -vt files I supplied, under the columns "Trimpoints Used Used Trimmed Length"?Used Trimmed Length"?
I short, this is correct, as far as I know.
That seems to be the case with my data but I don't have access to the cluster where I performed the runs with Newbler so I couldn't confirm.
Do you know how exactly the Vs and vt work, i.e. does Newbler just check if the sequences in these files are wholly contained in the reads, or does it perform alignments accounting for gaps and indels?
In any case if Newbler thinks that the adaptor or contamination is on the middle of a read, will it split the read, or exclude it?
Sometimes the reads, as set by the trimming points, will have only three less base pairs than the original that I supposedly defined. Or will have both start and end trimming points equal, that is as if they were excluded. My guess is that this because of those parameters, but I was unsure.
As an aside, thanks for your blog on Newbler, it is really informative !
Sometimes the reads, as set by the trimming points, will have only three less base pairs than the original that I supposedly defined. Or will have both start and end trimming points equal, which means that reads have been excluded.
My guess is that this because of those parameters, but I was unsure.
As an aside note, thanks for your blog on Newbler, it is really informative !
I don't know exactly how vs and vt work (there is no descprition in the manual). With vt, newbler only looks at the beginning and end of each read to see if an adaptor is present (surely with a certain identity <100%) and removes only that part of the read. With vs, the whole read is matched and removed or not. If start and end trimming points are equal, the read has not been excluded. Excluded reads don't show up anymore in the TrimStatus file.
I think you just want to mess around with the trimming options in Newbler. Check out the manual for trimming options.
And then it doesn't quite make sense to make a new SFF file. SFF is just a large binary file that also has pyrosequencing information... you just want a set of reads. If so, then you just want a fastq file with trimmed reads as input for Newbler. I would do the following:
1) create fasta/qual files
sffinfo -s file.sff > file.sff.fna
sffinfo -q file.sff > file.sff.qual
2) Create a Fastq out of the files. For example with bioperl http://www.bioperl.org/wiki/Merging_separate_sequence_and_quality_files_to_FASTQ
3) Then, use the fastx toolkit to create a fastq file and trim it. Either the fastx trimmer or quality filter executable will do what you want.
I believe that Newbler will work with a fastq file.
Maybe I was not clear enough in my question. I know how to mess around with trimming options in Newbler, that's what I did. I created a new sff file containing new trimming points that I found more appropriate. Yes, Newbler may work with a fastq, but ideally you will want to use the sff file, since that way you will be using the flowgram data as well.
I already produced the data, my doubts only regard the way Newbler produces this particular output file, namely what it considers as Used trimming points, and what is considered as the Original trimming points.
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