Spiking phiX into multiplexed libraries as well as barcode sequences

[cmawhinney]

Hi all, I have a two part question:

I am wondering if anyone here has tried spiking phiX into their multiplexed library samples? I heard from a couple people (as well as an Illumina rep) that this is a good way to ensure good base distribution to help with the cluster separation and identification that occurs for the 1st two bases (1st four if you have the new pipeline.

I am also wondering if anyone is multiplexing on a regular basis and has a good method for designing their barcodes? I have read all the prior posts on barcoding but the Cronn and Craig papers are almost 2 years old so I just thought I'd see if people were doing anything else to ensure good base distribution as well as avoiding any issues that might occur with mismatches.

To give you a little background we're looking to multiplex Chip-Seq samples, about 10 samples per multiplexed lane.

Thanks!

[clpX]

Hi all,

we are about to order 12 self made Illumina adapters with barcodes inserted at the position used by Craig et al., 2008. Here are my criteria for choosing the barcodes:

• 5 nucleotides long
• all 4 different nucleotides should be included
• indices differ at 3 or more positions
• no significant change in secondary structure of original oligos by index introduction

With such restrictions I could find a set of 12 indices but figured out that one needs to use 6 bp if one needs much more than 12. I used the sequences for the paired end adapters since they could be used for single read and paired end read.

We do ChIP-Seq as well and since we work with bacteria multiplexing is a nice option to reduce the cost.

[SeqR&D]

Hi clpX,
Could you share the protocol you used for ChIP-library preparation? I am have some trouble with my ChIP-libraries. The main failure in my prep is that I do not get amplification after size selection. I could actually see DNA on the gel when I cut out the 180-300 bp slice. However, after PCR all I got was just primer dimer that migrates way below 100 bp.
Thanks!

I would double check your primers compared to what you are putting on as adapters. If you have material, but you cannot amplify it...there is a simple reason.

1. Primers do not match/hybridize to target DNA...why this is happening is the game you will have to play. Either primers or adapters. Do you run a positive control for your PCR reactions? You should. Could be your Master Mix too.

[fkrueger]

Hi cmawhinney,

according to our experience you are only likely to loose (up to a quite substantial amount of) clusters if you are using a low number of multi-plex tags. We also performed a cluster density - low complexity sequence simulation program which supports our empirical findings that if you have 4 or more different sequence tags at the start of your sequences you should not run into any problems. Using 10 different barcodes should definitely provide enough variability in the start, thus spiking in a PhiX sample is not necessary.

If you have any further questions about initial biased sequences feel free to send me an email, by now we have done quite a few rescues of runs which failed due to this reason.