New contamination analysis suggests alternate strategy

Yesterday's post considered the factors affecting our current strategy for removing the contamination Rd-derived reads from our 'uptake' samples.  Since then I've looked at the read-mapping results using our new perfected reference sequences.  The problem of reads that have no SNPs and thus map ambiguously to both Rd and NP/GG is worse than we thought.  Below I'll describe this data and the new strategy I want to consider.The former post-doc sent us a spreadsheet with…

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Coverage and contamination in our DNA-uptake dataset

We finally have the corrected reference genome sequences for our DNA uptake deep-sequencing data.  The genome sequences from NCBI had errors and polymorphisms relative to the specific strains we used in the experiments, so the former post-doc used the sequence data from this experiment to create reference sequences that perfectly match the DNAs we used.The bright-coloured table below shows 8 of our 16 samples.  These are the samples that examined uptake of DNA from strain…

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Coverage and contamination in our DNA-uptake dataset

We finally have the corrected reference genome sequences for our DNA uptake deep-sequencing data.  The genome sequences from NCBI had errors and polymorphisms relative to the specific strains we used in the experiments, so the former post-doc used the sequence data from this experiment to create reference sequences that perfectly match the DNAs we used.The bright-coloured table below shows 8 of our 16 samples.  These are the samples that examined uptake of DNA from strain…

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Does expression of the toxA operon depend on ToxT as well as ToxA?

Short answer:  Yes, but not in the way we expected.First, here's a diagram showing the toxTA operon and the mutants we're examining:The grey bars show the extents of the deletions.  The ∆toxT and ∆toxTA mutants have a SpcR/strR cassette inserted at the deletion point, but the ∆toxA mutant has only a short 'scar' sequence at the deletion point.A few months ago I wrote a post about evidence that ToxA prevents transcription of the toxTA operon…

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Does expression of the toxA operon depend on ToxT as well as ToxA?

Short answer:  Yes, but not in the way we expected.First, here's a diagram showing the toxTA operon and the mutants we're examining:The grey bars show the extents of the deletions.  The ∆toxT and ∆toxTA mutants have a SpcR/strR cassette inserted at the deletion point, but the ∆toxA mutant has only a short 'scar' sequence at the deletion point.A few months ago I wrote a post about evidence that ToxA prevents transcription of the toxTA operon…

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Learning to use the NCBI Gene Expression Omnibus

As part of our workup for the toxin/antitoxin manuscript, I want to find expression data for the homologs of the Haemophilus influenzae toxin and antitoxin genes.  The former post-doc recommends that I use NCBI's Gene Expression Omnibus ('GEO') for this.I'll need to learn how to search the GEO for specific accession data and data from specific taxa.I'll also need to find out the specific identifiers for the genes I'm interested in, in the species I'm…

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Learning to use the NCBI Gene Expression Omnibus

As part of our workup for the toxin/antitoxin manuscript, I want to find expression data for the homologs of the Haemophilus influenzae toxin and antitoxin genes.  The former post-doc recommends that I use NCBI's Gene Expression Omnibus ('GEO') for this.I'll need to learn how to search the GEO for specific accession data and data from specific taxa.I'll also need to find out the specific identifiers for the genes I'm interested in, in the species I'm…

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How do non-competence genes respond to competence inducting treatment?

For the RNAseq part of the toxin-antitoxin paper, we should describe what we learn about how transfer to the competence-inducing starvation medium MIV affects genes not known to be involved in competence.The former undergrad left us with a set of Edge and DEseq2 analyses of changes in gene expression.  I discussed them here last summer (http://rrresearch.fieldofscience.com/2016/08/making-sense-of-rna-seq-comparisons.html).  Unfortunately I don't know how to properly interpret them.  The former post-doc suggested some analyses, but I'm reluctant to…

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How do non-competence genes respond to competence inducting treatment?

For the RNAseq part of the toxin-antitoxin paper, we should describe what we learn about how transfer to the competence-inducing starvation medium MIV affects genes not known to be involved in competence.The former undergrad left us with a set of Edge and DEseq2 analyses of changes in gene expression.  I discussed them here last summer (http://rrresearch.fieldofscience.com/2016/08/making-sense-of-rna-seq-comparisons.html).  Unfortunately I don't know how to properly interpret them.  The former post-doc suggested some analyses, but I'm reluctant to…

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What we learned from the RNAseq data: Part 1

So, we did a massive RNAseq study of competence, with 124 samples of H. influenzae cultures at different growth stages, in the rich medium sBHI and the competence-inducing medium MIV, and with wildtype or mutant genes affecting competence.  (You can use the Search box to find all the previous posts about this work...)  Here I specifically want to think about what we learned about competence from the competence-induced cultures (cells transferred from sBHI to MIV).…

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