What’s noise, what’s Illumina bias, and what’s signal?

The PhD student and I are trying to pin down the sources of variation in our sequencing coverage. It's critical that we understand this, because position-specific differences in coverage are how we are measuring differences in DNA uptake by competent bacteria.Tl;dr:  We see extensive and unexpected short-scale variation in coverage levels in both RNA-seq and DNA-based sequencing. Can anyone point us to resources that might explain this?I'm going to start not with our DNA-uptake data…

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Do the rpoD hypercompetence mutations eliminate the normal diauxic shift?

I've been going over the RNA-seq data for our rpoD1 hypercompetence mutant, looking for changes in gene expression that might help us understand why the mutation causes induction of the competence genes in rich medium.Here's a graph showing the results of DESeq2 analysis of the expression differences between the wildtype strain KW20 and rpoD1 cells at timepoints B1 and B2.  B1 is true log phase growth in rich medium; OD = 0.02.  B2 is OD…

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Analysis of NP-GG differences (I can’t help myself!)

Despite my sensible conclusion to the previous post, I've rushed in with a bit of analysis of the reasons for the differences between the NP and GG uptake-ratio peaks.I was able to do this because the PhD student just posted two new graphs, showing the uptake peaks in syntenic 20 kb segments of the NP and GG genomes. The peaks for the two genomes are in the same places because the underlying DNA sequences are…

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Unexpected differences in uptake of DNA from two closely related strains

The PhD student's long careful reanalysis of the DNA uptake data has finally produced uptake ratio plots.  These confirm a surprising difference between the DNAs from two closely related strains, 86-028NP ('NP') and PittGG ('GG').  We also saw this difference in our preliminary analysis, but we thought it might be an artefact of how the analysis was done.In the experiment underlying this data, cells of a third strain, KW20, took up DNA that had been…

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How many contamination-control replicates can we do?

This is a continuation of the previous post.For each of our 12 genuinely contaminated uptake samples we want to create multiple replicate fake-contaminated input samples, each fake-contaminated with an independent set of Rd reads at that sample's level of contamination.For example, our UP01 sample has 5.3% Rd contamination.  Its corresponding input sample is UP13.  UP13 has about 2.7 x10^6 reads, so to make a fake-contaminated sample for UP13 we need to add (2.7x10^6 * 0.053)/(1-0.053)…

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Almost there: making the uptake ratio graphs

Yesterday the PhD student showed me the results of his contamination-correction tests.  They confirmed that our new error-correction strategy works, and suggested an improvement.The problem and the strategy:  We want to know how efficiently different segments of NP or GG DNA are taken up by competent Rd cells.  All of our 12 'uptake' samples are contaminated, consisting of mostly reads of NP or GG DNA taken up by Rd cells plus varying amounts of contaminating…

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UBC’s Faculty Pension Plan, for dummies

Yesterday I went to an information session about UBC's Faculty Pension Plan, designed for faculty approaching the age when we have to decide what to do with the money the Plan has accumulated on our behalf.  Even if we choose not to retire (or not yet), by age 71 contributions to the Plan will end and we must make a decision.Until retirement or age 71, the Plan has been taking money from our salaries and…

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UBC’s Faculty Pension Plan, for dummies

Yesterday I went to an information session about UBC's Faculty Pension Plan, designed for faculty approaching the age when we have to decide what to do with the money the Plan has accumulated on our behalf.  Even if we choose not to retire (or not yet), by age 71 contributions to the Plan will end and we must make a decision.Until retirement or age 71, the Plan has been taking money from our salaries and…

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Ways to test contamination control

In the previous post I proposed an alternative method to control for contaminating recipient DNA in our donor DNA uptake samples.  Because we've never done this before (and probably nobody else has either), we need ways to check that it accomplishes what we want.Here's one way: We already have made samples of pure donor DNA reads (from strain NP or GG) that have been deliberately contaminated with reads from the recipient Rd (10% Rd, 90%…

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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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