454 MIDs

[JackieBadger]

Usually, when you get back data from a multiplexed run, the MIDs are already removed from the SFF file (better: offsets are shifted by the length of the MIDs used).
So if you extract your reads with e.g. 'sffinfo' you get the "clipped" sequence (unless your are using the flag '-n'). Your fasta files do not contain the MIDs anymore.
You should keep this in mind, also when using other tools for extraction.
What tool have you been using for sff->fasta extraction?

cheers,
Sven

If a sequencing company removed the MIDs which I had attached to ID individuals, I wouldn't pay them. The whole point of MIDs is so they can be used to sort sequences.
I have never had MIDs removed from my data, only 454 adapter sequences.

You may as well parse the data using your FASTA and QUAL files. These will have the MIDs. If your sequences do not contain MIDs, you either didn't ligate them properly or the sequencing company shouldn't be paid. I highly doubt they would remove MIDs.

[sklages]

Usually, when you get back data from a multiplexed run, the MIDs are already removed from the SFF file (better: offsets are shifted by the length of the MIDs used).
So if you extract your reads with e.g. 'sffinfo' you get the "clipped" sequence (unless your are using the flag '-n'). Your fasta files do not contain the MIDs anymore.
You should keep this in mind, also when using other tools for extraction.
What tool have you been using for sff->fasta extraction?

cheers,
Sven

[JackieBadger]

Hi everyone. I've searched everywhere but haven't quite found the solution to my problem so bare with me if I'm asking a simple question.

I just got back from 454 data that we generated using the Fluidigm access array. I am having difficulties parsing out the MIDs. I've converted the .sff files to fasta files and tried parsing out the MIDs with fastx tools. For some reason, the barcode splitter option is not working. I'm sure my syntax is correct so I think something is wrong with my fasta file. I know the keytags and adapters are still on my sequences. Is this the problem? I'm not sure what I should do. We are not big fans of Roche's software especially AVA so we are trying to find a different solution. Also, have people been successful using novoalign for mapping 454 data?


Any information is greatly appreciated!!!!! Thank you!

Ali

Hey,

Yep unless you have code to do this it isnt easy (what with you perhaps having a bunch of sorting parameters you may be interested in).

Geneious, offers a free trial, is easy to use, and super cheap for students. You can do it there. http://www.geneious.com/


I used jMHC, to parse mine as I found their parsing criteria particularly stringent (No Ns in primers or sequence, and 1bp = new allele) http://code.google.com/p/jmhc/

I found that with jMHC parsing could take hours, despite running on a VERY powserful desktop PC. Ran it on my Mac laptop -wizzed through it in minutes!

I attempted to get SESAME up and running, but it is a fiddly process and got tired of trouble shooting... http://bioinformatics.oxfordjournals...2/277.abstract

Good luck!

J

[aligenie]

Hi everyone. I've searched everywhere but haven't quite found the solution to my problem so bare with me if I'm asking a simple question.

I just got back from 454 data that we generated using the Fluidigm access array. I am having difficulties parsing out the MIDs. I've converted the .sff files to fasta files and tried parsing out the MIDs with fastx tools. For some reason, the barcode splitter option is not working. I'm sure my syntax is correct so I think something is wrong with my fasta file. I know the keytags and adapters are still on my sequences. Is this the problem? I'm not sure what I should do. We are not big fans of Roche's software especially AVA so we are trying to find a different solution. Also, have people been successful using novoalign for mapping 454 data?


Any information is greatly appreciated!!!!! Thank you!

Ali