Second barcode set

There is now a second set of barcoded adapters that allows higher multiplexing. They also appear to address the quality issues which have been observed in the second read of GBS runs.

This blog post has 1) Info on how to use the barcodes and where they are and 2) some data that might convince you to use them.


These add a second barcode to the start of the second read before the MSP RE site. The first bases of the second read contain the barcode, just like with the first read. Marco T. designed and ordered these and the info needed to order them is here:

I’ve labeled them MTC1-12 and the barcode sequences are as follows.


They are used in place of the common adapter in the standard protocol (1ul/sample). One possible use, and simplest to use as an example, would be to use these to run 12 plates in a lane. In this case you would make a master mix for the ligation of each plate which contains a different MTC adapter.

Where are they? In the -20 at the back left corner of the bay on the bottom shelf in a box that has a pink lab tape label that says something to the extent of “barcodes + barcoded adapters 1-12”. This contains the working concentration for each of the MTC adapters. Beside that is a box containing the unannealed and as ordered oligos and the annealed stock. The information regarding what I did and what is in the box is written there. The stock needs an additional 1/20 dilution to get to the working concentration

How it looks

First, the quality of the second read is just about as nice as the first read. Using fastqc to look at 4million reads of some random run:

Read one:

Read two:

Now, for the slightly more idiosyncratic part: read counts. In short I dont see any obvious issue with any of these barcodes. I did 5 sets of 5 plates/lane. For all the plates I used the 97-192 bacodes for the Pst side. Then each plate got a differnt MTC barcode for the MSP side. Following the PCR I pooled all of the samples from the plate and quantified. Each plate had a different number of samples which I took into account during the pooling step. Here is the read counts from a randomly selected 4 million reads corrected to number of samples in that plate. Like I said it is a little idiosyncratic but the take home is that they are about as even as you might expect given usual in accuracies in the lab, my hands, and the fact that this is a relatively small sample.

Lane 1
MTC5	14464
MTC1	13518
MTC7	14463
MTC9	13448
MTC3	14232

Lane 2	
MTC10	30395
MTC6	11267
MTC2	8263
MTC4	19295
MTC8	14766

Lane 3	
MTC5	16631
MTC7	17315
MTC11	11623
MTC9	16256
MTC3	13831

Lane 4		
MTC10	11302
MTC6	12120
MTC4	10326
MTC12	18959
MTC8	12832
Lane 5
MTC1	13151
MTC6	13490
MTC2	12851
MTC11	12460
MTC12	17296

Paired End for Stacks and UNEAK

Both stacks and uneak are made for single end reads. If you have paired end data here is a little cheat that puts “fake” barcodes onto the mate pairs and prints them all out to one file. It also adds the corresponding fake quality scores.

perl BarcodeFile R1.fastq R2.fastq Re-barcoded.fastq

BarcodeFile should look like (same as for my demultiplexing script) spaces must be tabs:
sample1 ATCAC
sample2 TGCT

# note this could also look like this:

As it does not actually use the names (it just looks at the second column).

Here it is:

CheapEasy DIY Barcodes in R

I couldn’t believe how expensive the software was for writing barcodes, so I wrote a short program in R to do it for FREE. And, frankly it should be faster and easier if you already have your labels in an Excel file. You don’t really need to understand the program or even R functions to use it, as long as you know how to run an R program.

Setup and Overview:

[UPDATED (see notes below)] – R-code. Start with this (Note I could not upload a .R file, so this is .txt but still an R program).

Input – barcodes128.csv – You need this file to run the program. Save it in your working directory (see comments in R code for how to set this). AND labels.csv – This is a sample file showing the format for your labels. Even though it’s a .csv, it is a single column with each label as a separate row, so there are no actual commas

Output – BarcodesOut.pdf – A sample output: a pdf file for the 0.5″x1.75″ Worth Poly Label WP0517 (Polyester Label Stock), currently in the lab

That’s really all you need to know, everything that follows is extraneous info. If you have any problems, check out the Detailed Instructions, Troubleshooting Tips, or add a comment below. Continue reading

Illumina Sequencing Adapters and Barcodes (Dan E.)

As of March 2012 we are using the Bioo Scientific NEXTflex barcoded adapters for WGS sequencing libraries made by ourselves, (well me so far). The set we are currently using comprises 48 barcodes, so we can multiplex up to a 48-plex in one lane on the Illumina HiSeq sequencer.

Bioo Sci. 48 barcoded adapters

Below are the sequences of the Illumina adapters and the 48 barcodes we are currently using. Continue reading