Friday, March 20, 2015

Planning more RNAseq runs

We need to decide what additional RNAseq runs we're going to do, given the papers we'd like to write and the limited resources we have.  This post attempts to clarify the issues, following on a long discussion with Josh about this yesterday afternoon.

Here's a screenshot of the latest version of the table showing both what we've done and the runs we might still do.

We anticipate using this data in several papers:

1. A paper about the toxin/antitoxin genes (authors Hailey, Rosie, Josh, Sunita, Scott?):  This paper will consider the gene expression data, the phylogeny, and the phenotype of knockouts in H. influenzae and A. pleuropneumoniae.  For it we will directly need three MIV time-course replicates of KW20, and the toxin, antitoxin and double knockout strains (toxx, antx, taxx in the table above).  We will also need to have identified the set of Sxy-dependent competence-induced genes, which will use the three replicates of the sxy-knockout time-course (sxyx).  We can control for any effects of the spcR cassettes in the 'marked' strains by analyzing data we have.

We therefore need to still do:

  • 2 replicates of the antx (unmarked) MIV time-courses to replace those that t=urned out to be the comN knockout (cmnx).
  • At least 1 replicate of the toxx (unmarked) MIV time course, to complement the 1 unmarked and 2 marked we already have.

2. A paper about the role of hfq in competence (authors Scott, Rosie, Josh, Sunita?):  This paper will consider the phenotype of the hfq knockout and its effect on gene expression and cAMP levels.  For it we will need the three KW20 MIV time courses, the 3 sxyx time courses to identify Sxy-dependent genes, and 2 crpx and hfqx MIV time-courses. We need to sort out ow cAMP levels change - Scott has done beta-gal assays. We may also need to identify the small RNAs that Hfq might interact with, unless this information is available from published hfq or other papers.

We therefore still need to do:

  • At least 1 replicate of the hfqx MIV time-course
  • At least 1 replicate of the crpx MIV time-course
  • Maybe 2 replicates of one or more small-RNA samples (RNAs prepared with Trizol rather than with Qiagen RNeasy columns).

3. A paper about the rpoD hypercompetence mutation (authors Lauri, Rosie, Josh?): This paper will report the identification of a hypercompetence-causing point mutation in rpoD (maybe more mutations if Lauri's work pans out).  In the rpoD mutant in sBHI, sxy RNA is up 1.6-fold at B1, 3.3-fold at B2, and 1.8-fold at B3. We will definitely need another replicate of these samples.  Lauri's analysis of sxy-beta-gal promoter fusions also found that sxy transcription is up in this mutant.  There aren't very many dramatic changes in other genes, and without any replication it's very hard to tell what might be significant.

We need:

  • at least one replicate of the rpod sBHI samples

4. A paper about identification of HI1457/HI1456 as a competence-induced operon. (authors ???; this result might be in the same paper as 3): These RNAs are up strongly in all three hypercompetence mutants in sBHI, and 1.6-2-fold in wildtype cells in MIV.  HI1457 (291 aa) is tagged as 'opacity protein' and HI1456 (179 aa) as 'hypothetical protein'. (Note to self: this is not Hanna Tomb's por gene (dsbA homolog? HI0846).)  Are they CRP-S regulated?  They are way down (8-12-fold) in all the crpx MIV time-course samples, including the M0 sample, which is OD=0.2 in sBHI.  But, oddly, they are down only 1.6-2-fold in the other crpx samples in sBHI (OD=0.02 and OD=0.6) and in the sxy knockout in sBHI and MIV.

5. A paper about regulation of competence (or as part of one of the other papers):  Levels of sxy RNA are up 2-3 fold (highly significant) in the sxy1 hypercompetent mutant samples in sBHI (1.8-fold in B1, 3.1-fold in B2, and 2.7-fold in B3).  This is unexpected under the model that these mutations just affect translatability.  In the KW20 MIV time-courses, sxy RNA is up 11.9-fold at M1, 31.2-fold at M2, and 5.1-fold at M3.

6. A paper about the murE749 hypercompetence mutant: (or as part of one of the other papers):  In the murE749 mutant in sBHI, sxy RNA is up 2.2-fold at B1, 3.1-fold at B2, and 2.1-fold at B3.  These increases aren't significantly higher than those in the rpoD mutant, but the increase in competence is much more dramatic (100-fold more than rpoD753, 1000-or-more higher than KW20).  So the difference in competence between murE749 and rpoD753 must be due to something other than the increase in sxy RNA.  We have two replicates of the sBHI samples - another would be nice but isn't a very high priority.

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