Sequencings of pooled BAC clones without Barcoding

Question

[gangireddy]

Hi people,

I am trying to assemble pacbio reads from RS II. But, the problem is that

1) we have pooled 6 BAC clones per cell but we have not barcoded the Samples. So, my first question is that can these reads be assembled.

2) The second problem with same data is we pooled the clones thinking that inserts may be overlapping sequences which seems to be true has resulted which resulted some regions overlapping high very high coverage and some regions at end and non overlapping having low coverage. Does anybody know how to tackle this problem.

[gangireddy]

Sorry, forgot to mention Thanks in advance

Answer 1

[GenoMax]

Number 1 may have been possible if there were no overlaps between the BAC's.

But question number 2 makes it sound like that is NOT the case. So I have a doubt that you are going to get six independent assembled BAC's but minimally you should be able to get the regions that are non-overlaping as multiple contigs with just the data that you have. If you are lucky and if some of the pacbio reads start/end in such a way that they internally contain the overlapping regions then you may be able to do much better.

You are going to have to try and do the assembly to see what happens.

[gangireddy]

Thanks for the reply.

I have tried assembly using HGAP protocol, celera assembler (v 8.1 individually) and I have also tried canu.

But in all cases I was left with more than 10 spurious assembled contigs. None of them are what I need, as some of the markers are present while some other markers are absent in the assembled contigs. Thereby, conveying that none of the contigs are useful and all of them are spurious.

[GenoMax]

It may be possible to tweak the HGAP alignment parameters to see if you could do better. Are these BAC's overlapping much more than you had originally thought? If that is the case then this may be a lost cause (if you have the libraries you could run them individually). In any case I suggest that you try contacting PacBio tech support to see if they can help with HGAP settings.

[gangireddy]

Thanks again for the reply,

I have already played with the alignment parameters. the assembly of around 10 to 20 contigs of the spurious assembly is the best i have achieved till now after many runs.

[GenoMax]

You may have to bite the bullet and re-sequence the libraries independently (short of remaking them with barcodes).

[gangireddy]

Thank for advice. I was also thinking the same thing. But I was not confident enough to go through with it as it is costly. Also, I was hoping there might be some other way.

Answer 2

[rhall]

It's going to be difficult to assemble the BACs due to the overlaps, but it still may be possible. It's probably not simply a matter of HGAP settings, it will likely involve manual tweaking. You can email me directly and we can discuss in detail [e-mail]

[gangireddy]

Thanks. I'll definitely mail you.