[ScottC]

Amplicons of 1-1.5 kb should not be a challenge for Nextera, as we have gone as low as a few hundred bp

Won't it have to be only a few hundred bases for Illumina library preps?
Also, do you have any idea about the release date for this product?

Scott.

[Lestag]

Scott, you're correct about the final tagged fragment length, but the process allows simultaneous fragmentation and tagging from a range of original DNA sizes. Remember that a modified transposome is breaks the input DNA and insert tags.
Release will be soon...

[ScottC]

Hi Lestag,

Yes, I understand how it works... I just misread your statement! For some reason, I thought you were saying that you'd fragmented larger DNA to result in 1.5kb fragments at the end of the process!

Having re-read it, I now see that you were talking about 1.5kb starting material size, not the final 'tagmented' size range.

Cheers,

Scott.

[berthold]

Perl script available for "digesting" fastq files?

Hi,

I'd like to come back to this thread. I've done it - mixed a lot of different PCR reactions (with different primers), and had them co-ligated, fragmented and Illumina-sequenced by a service provider. The reads now match nicely to PCR template references, but because of the chimeric reads (I guess), coverage drops at the starts and ends of each fragment.

I thought it should be possible to tweak a Perl script that creates restriction fragments for the purpose of splitting up the chimeric reads, because the co-ligation joints, made up of 2 primer sequences each, resemble restriction sites in a way. I am new to Perl too - does anybody have a script that "digests" fastq files (creates fastq files containing the restrition fragments)? I guess I could feed my own primer sequences into such a script, but I don't think I'll be able to write it from scratch. I am aware such a thing may run for a while, but I'd like to give it a try.

grateful for any help - cheers,
Berthold

[sci_guy]

Hi Berthold,

At least three ways to do it.
1) Write a Perl script to remove exact matches. Parse the FASTQ, and look for the primer in each read with regular expressions. Trim the reads with hits using the regular expression result. Very fast run time.
2) Write a Perl script to remove inexact matches. As above with an extra check on reads without exact matches for inexact matches. This will run much slower. Can use an approximate match library or BioPerl and perform alignments to your primer. Considerably trickier to write for a novice programmer.
3) Upload the FASTQ data into Galaxy. Under the "NGS: QC and manipulation" section use the "Clip adapter sequences" tool. Too easy.

If you take the trickier programming route I have some scripts in Perl and/or Python that I could send to get you started.

Cheers,
Jason.