control data

[mingkunli]

I don't do wet work. What I can tell you is they tried lots of primers, and most of them don't work, even works, only on some individuals.
concerning the barcoding, we use the protocol published on nature protocol 2008 which is for 454, meanwhile, we are designing new protocol.

[vasvale]

would it be possible to have some details about how you do long range PCR and barcoding? I noticed that there are so many polymorphisms that it seems hard to design primers in conserved regions

thanks

Vasvale

[bioinfosm]

Yes, we used long range pcr and have also bar-coded the samples.

[vasvale]

mtDNA

Thank you, my question is about the upstream phase of the experiments, do you enrich by PCR pr long range PCR? can you append barcodes?

thanks

Vasvale

[basickler]

I use the following for mt when aligning against the human NCBI ref and it seems to do the job.

1234678:PAIR_PARAMS --circular=cMT.fa --min-single-read-alignment-score=1

If you don't have paired end you could probably do something a bit brute force and create two genome files, one normal and one with the beginning offset a few hundred base pairs (cut a bit of the end and move it to the beginning). Then a quick script should be able to correct and combine the two. Or you could use a different aligner than eland.

Brad

[vasvale]

mitochondria

Hello

can you kindly let me know how you proceed to seq mtDNA? are there papers?
thanks

Vasvale

[bioinfosm]

That's strange if it's not the overlap region of your long-range pcr product(in case you use long range pcr).

Bingo. Thats what I determined as well

[mingkunli]

That's strange if it's not the overlap region of your long-range pcr product(in case you use long range pcr).

[bioinfosm]

Despite not worrying about the cut in circular mt genome, I get peaks of coverage at the end points of linearized sequence I use (cambridge reference)

[mingkunli]

hi seq_GA

thank you for remind me that, but seems it only works with paired reads, unfortunately, I just have single end data

[seq_GA]

Eland has an option

8:PAIR_PARAMS --circular

can be used for mitochondria as well as circular bacterial genomes.

Please let me know the advantage of using above said.
Regards

[mingkunli]

we are also thinking that

[bioinfosm]

I have been doing MT alignments and taking the ref seq as is, ignoring missed reads at the ends, and still seem to get useful results.

One option could be to add the first 10-20 bases to the end, making sure the falling-off reads align there..

[mingkunli]

Now I have my first Solexa data, as it's for MT, which is circular, therefore, some of the reads will overlap the begining and ending, is there some software that can do local alignment, which means, part of the read aligns to the begining and other aligns to the ending?