Heterozygosity, read counts and GBS: PART 2

Subtitle: This time… its correlated.

Previously I showed that with the default ML snp calling on GBS data, heterozygosity was higher with high and low amounts of data. I then took my data, fed it through a snp-recaller which looks for sites that were called as homozygous but had at least 5 reads that matched another possible base at that position (i.e. a base that had been called there in another sample). I pulled all that data together, and put it into a single table with all samples where I filtered by:

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GBS, coverage and heterozygosity

I’m running some tests on my GBS data to look for population expansion. I know from looking at GBS data from an F1 genetic mapping population that for GBS data heterozygotes can be under called due to variation in amplification and digestions. Also, for my data observed heterozygosity is almost always under expected. Heterozygotes can also be overcalled when duplicated loci are aligned together. The tests I’m going to use explicitly use observed heterozygosity so this is worrying.

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GBS protocol Version 2.0

Hi all,

Here is the long awaited updated GBS protocol.

PROTOCOL ->>>> GBSv2.0

There are three main changes from the previous protocol.

-After digestion and ligation, all the product is kept so more attempts can be made at the PCR.

-The PCR uses Phusion Taq, has longer extension times and one additional cycle with more primers/Taq.

-Size selection is done using AMPure beads instead of gel extraction.

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GBS multiplexing

GBS_mutliplexing contains 2 scripts that may be useful to you if you are using GBS data. One essentially formats GBS reads for Tassel. The other demultiplexes the reads. A readme file in there explains things in more detail.

Edited: edited the readme and added a script to convert qseq to fastq

GBS Protocol (GregO)

Kristin and I have been working on GBS for a long time and since it now seems to be working, we wrote up a protocol. It is mostly the same from Greg Baute’s previous protocol, but with a few key changes (More DNA, more PCR). I’ve made it look nice and included a diagram for ease of thought.

Also, the official pronunciation of GBS is ‘jibs’

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STACKS installation (Rose)

Installing stacks on Ubuntu Natty Narwhal or Oneiric Ocelot

STACKS is a piece of software produced by Julian Catchen in the Cresko lab. It’s designed to identify loci and alleles from RAD (or GBS) reads either de novo or after alignment to a reference. It consists of several modules that can be run separately, but to completely install it as a pipeline, it relies on a web server, unfortunately. Many of the required instructions are given in the README file, but because nobody in our lab is an expert on this, we had to fiddle around to get the program running on our Ubuntu machines.

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