TruSeq Indexing

[felvis56]

Dear all,

this is my first post so be kind. I have a stupid question but after days of reading Illumina protocols my brain has ceased to function.

I have 24 samples which I wish to run through the MiSeq, I have a Truseq kit with 12 indices and I am using the Low Throughput protocol which suggests a single index, how do I index 24 samples?

I have previously used the Nextera kit with i5 and i7 indices which made sense but I'm not sure I get how a single index works.

Thanks in advance.

[mastal]

TruSeq Indexing

If you only have 12 different barcodes for 1 index, then you can only pool 12 samples for each run on the MiSeq, so you would have to do 2 runs with 12 samples in each.

[felvis56]

Thanks. I thought this was probably the case and when it states 48 reactions it just meant there were enough supplied to do 4 runs of 12 but I had hoped that there was a clever way to multiplex. Oh well.

[kcchan]

The TruSeq LT and HT reagent kits have different adapter sequences. The LT kits have a single index, while the HT kits have dual indices. There are two LT kits, each with a set of 12 barcodes for a total of 24. The HT kit comes in a plate of 96 unique adapter combinations. This is different from Nextera where the adapters are provided in 20 tubes for you to mix and match.