Barcode sequences

[NJD]

Hi folks,

I’m working on multiplexing using barcoded adapters – the non-Illumina version where barcodes are read as the 5’ end of the fragment. From various discussions in these forums it sounds like a number of groups are doing this routinely, and with good success. I would like to pool up to 96 samples in a lane. Would anyone multiplexing at this level be willing to share their barcode sequences?

I am, of course, also considering just designing my own… any suggestions on particular sequence patterns to avoid?

Thanks!

Nathan

[tujchl]

96 samples in one lane? Are you kidding me?
what kinds of work do you want to do? theoretically you can mix 96 samples and sequence them in one lane. but the deep of you sequencing can not be very well even very bad. you can even get nothing usefully from your experiments

[nilshomer]

96 samples in one lane? Are you kidding me?
what kinds of work do you want to do? theoretically you can mix 96 samples and sequence them in one lane. but the deep of you sequencing can not be very well even very bad. you can even get nothing usefully from your experiments

96 pooled samples would be great for many reasons not limited to assessing the frequency of a mutation in a gene (or genes) across many samples.

[tujchl]

I afraid that sequencing 96 samples in one lane would not guarantee you find mutations and gene what you say because of sequencing depth

[nilshomer]

I afraid that sequencing 96 samples in one lane would not guarantee you find mutations and gene what you say because of sequencing depth

@tujchl

Lets say you produce 20M 75bp paired end reads from one lane. That equals 3 billion bases, or approximately 30 million bases per individual. Therefore you could cover a 100Kb region at 300X coverage. Of course, you may not align all the reads, but still that that is sufficient coverage if barcodes are used.

I am not understanding your justification.

@NJD

I would put in some redundancy for your barcodes. You need a minimum of four bases to barcode 96 people, and you could use a fifth base to detect an error in any of the previous four etc. With longer reads, the extra base is not that big of a hit.