Pro-strains sequencing completed

[Atina Cote]

Hi - the sequencing of the pro-Donors (?) is complete and the data is on Basespace.  Joe, will you take care of the analysis? 

Thanks,
Atina

[Joe Mellor]

Yes, I will download the data and get to it over the weekend.

Joe

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[Paul Bansal]

Thanks Joe.

Attached is a spreadsheet with how I set up the indices for the Reamp.

Let me know if you need any more info.

Best,

Paul

[Javier Diaz]

Hi Joe,

A few strains (25) in the files 'Pro*_Pick_List_20131115.txt' you sent us on 15Nov2013 show 'GOOD' status for the LoxP and Priming sequences in the Google doc you shared with us (hence they passed the QC), but their barcode sequences show one or more undefined bases, e.g:
ProD_005_A02    A-TTGCCTCCGGCATCTAAG (UPtag)
ProR_017_C12    ACCGTGGGC-AGGTTTTCCA (UPtag)
ProR_009_B09    TCTGCGTGGCAC-CTTCTCT (DNtag)

I could map 18 out of the 25 in the paired-end reads I got from the Neutral Double mutants (generated in array format) and found that in all cases they are missing either a 'C' at the end, or a 'G' at the beginning, rather than a gap in the middle.
The above examples look like:
ProD_005_A02    ATTGCCTCCGGCATCTAAGC
ProR_017_C12    ACCGTGGGCAGGTTTTCCAC
ProR_009_B09    GTCTGCGTGGCACCTTCTCT

I looked at the extra 5bp in the 25bp/25bp paired-end reads and they match the expected Priming region, like:

ATTGCCTCCGGCATCTAAGC,CTCAG
ACCGTGGGCAGGTTTTCCAC,CCCTA

So I guess it's just a minor parsing issue of 'CC' or 'GG' (although I'm not sure why there are dashes in the middle).

Could you please take a look and see if we can correct it during your QC of the new batch of Pro* strains?

Attached is the full list of 25 strains.

Thanks.

J.

**********************************************************
J. Javier DIAZ-MEJIA
Postdoc at Donelly CCBR, University of Toronto.
Profr. Fritz Roth group (room 1040-B)
http://www.lusara.org/jdime
E-mail: javier [dot] diazmejia [at] gmail [dot] com
**********************************************************

[Joe Mellor]

Yes, this is relatively easy tune I think, and thanks for sending me the corrections.  I suspect the dashes are usually there because of how the SW alignment is made to the expected template sequence, i.e. primer-N20-primer.  If there are two possible alignments, one of which places a gap in the middle of the N20mer, or the other that places a mismatch at the terminal base of the primer, I think the SW scoring matrix is set to prefer putting a gap in the N20mer. I think I decided this was more likely because we expected it to be more likely that there would be truncation errors in the oligos used to make the construct than it was that there would be errors.  Does that make sense?  Anyway, I will take a closer look at this also when generating the list of good ProDonors.

Joe

[Atina Cote]

Thanks Joe.  Is the batch 2 pro-recipient cherry pick list ready for us?

Atina

Atina Cote, PhD
Research Associate
Roth Lab
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto
Samuel Lunenfeld Research Institute, Mount Sinai Hospital