Arichard:Notebook/AR150914

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Revision as of 17:03, 17 September 2015 by >Andrew (Created page with "==Plan== ===Motivation=== We are using the VarBin method from CSHL (Baslan et al., Nature Protocols, 2012) to call CNVs in single nuclei. Briefly, artificially generated 36 ...")
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Plan

Motivation

We are using the VarBin method from CSHL (Baslan et al., Nature Protocols, 2012) to call CNVs in single nuclei. Briefly, artificially generated 36 base reads from every position of hg38 (~3 billion reads) were mapped back to hg38 using Bowtie (-n 2 -e 70 -m 1). Only uniquely aligning reads were retained (~85%); no multiple alignments were considered. Bin boundaries were defined such that all bins have equal expected coverage from a genome with no CNVs. We know that even under ideal conditions, a uniform genome will still display non-diploid copy numbers in certain bins, called "bad bins". These are largely due to incomplete assembly of repetitive regions, such that these regions appear unique in the reference. This leads to a systematic bias which can be profiled empirically and corrected.

Goal

Create a null distribution for every bin using bulk libraries from GM12878 such that a z-score can be calculated for single nuclei.

Protocol

Results

CNV plots

The noise from these libraries was higher than expected for bulk, but close to ideal from single cell. Need to verify that this data is indeed appropriate to make null distibutions.

File:BulkGM12878-A.all cnvPlot.png File:BulkGM12878-B.all cnvPlot.png File:BulkGM12878-C.all cnvPlot.png File:BulkGM12878-D.all cnvPlot.png File:BulkGM12878-E.all cnvPlot.png File:BulkGM12878-F.all cnvPlot.png File:BulkGM12878-G.all cnvPlot.png File:BulkGM12878-H.all cnvPlot.png File:BulkGM12878-I.all cnvPlot.png File:BulkGM12878-J.all cnvPlot.png

MAPD plots

The noise trajectories are decreasing nicely; perhaps these libraries were somewhat under-sampled.

File:BulkGM12878-A.all mapdPlot.png File:BulkGM12878-B.all mapdPlot.png File:BulkGM12878-C.all mapdPlot.png File:BulkGM12878-D.all mapdPlot.png File:BulkGM12878-E.all mapdPlot.png File:BulkGM12878-F.all mapdPlot.png File:BulkGM12878-G.all mapdPlot.png File:BulkGM12878-H.all mapdPlot.png File:BulkGM12878-I.all mapdPlot.png File:BulkGM12878-J.all mapdPlot.png