How to generate barcodes for HiSeq

Question

[sebastianzeki0]

Hello. I want to run around 50 samples on a HiSeq lane. I have done this on MiSeq with success and am just scaling up. With the MiSeq I ran 11 samples with 11 barcodes as that's all I was given (they are custom primers). So now I need more barcodes (6 nucleotides). I'm sure I could generate a load of random barcodes but I want to make sure they don't cause havoc. Is there a best practise for barcode generation in this situation?

[Dan D]

Are you familiar with dual-indexing?

[sebastianzeki0]

No. But I guess what I'm asking is whether I can use a load of made up barcodes for single indexing or how I minimise confusion between barcodes when sequencing

Answer 1

[epistatic]

Index design and analysis software for Meyer M, Kircher M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb Protoc. 2010 Jun 1;2010(6) doi:pdb.prot5448. PubMed PMID: 20516186

https://bioinf.eva.mpg.de/multiplex/

1. Designing index sequences with some edit distance (create_index_sequences.py)
2. Selecting a balanced subset of indexes (suggest_subset.py)
3. Evaluating the channel/base composition of a defined set of indexes (eval_selection.py)

[Shicheng Guo]

So we don't need to buy it from Illumina, right? Can I design it and synthesize from routine nuclear acid primer company? will this cheaper than Illumina? What's the cost for 2048 barcodes? Thanks.