Maximum Sample per MiSeq Run

[Nikvailo]

Hi,

In both of MiSeq manual and Custom Amplicon Prep. Guide, it is said that maximum 96 samples can be read per run. It is seen that number of i5 and i7 primers (w/ indices) limits the number of samples.

There are 8 different i5 primers and 12 different i7 primers and combination of these create tags for 96 samples. Is it possible to design additional i5 or i7 primers (w/ custom indices) to increase sample number that can be read per run?

Thank you,

[IDDan]

We do MiSeq runs with 300+ amplicons per run. We are currently limited by primer cost, not the HiSeq hardware/software. The keys are (IMO):
1) having the bioinformatic support to sort out the reads when the run is done
2) properly designing barcodes/indexes/tags so that they are sufficiently difference from each other to believe custom sample barcodes (97+) wouldn't be mis-called as barcode 1-96 or (each other) when you are sorting out which reads go to which barcodes.

[JackieBadger]

It all depends on the depth you want to achieve per amplicon.
We run ~ 1000 samples per run achieving depths in the 1000s

[thomasblomquist]

We scale to 400-1000 samples for amplicon library prep for a flow cell lane on Hiseq and 50-200 samples for miseq easily. Just make sure your barcoding steps are clean and contamination free to prevent "bleed-over" of falsely indexed amplicon products between sample indices. IDT offers Trugrade NGS barcoding primers that work quite well and are worth the investment for zero cross-contamination of reagents. Our libraries are focused panels of 15 to ~100 amplicons per sample.

-Tom

[thomasblomquist]

Oh, and yes, you need custom bioinformatics/software pipeline to parse out the custom indexes for each sample. The 96 limit is probably Illumina's limit based on their own reagent kits.

-Tom

[james hadfield]

Dual-indexing should scale cheaply to 384, 1536 and beyond. Uising a pair of indexes to create some thing unique allows just 20 PCR primers to create the 96 used in Nextera for instance. Adding another 20 allows 384 indexes, and increasing to 80 primers (32x i5 & 48x i7) gets you to 1536.

[JackieBadger]

Oh, and yes, you need custom bioinformatics/software pipeline to parse out the custom indexes for each sample. The 96 limit is probably Illumina's limit based on their own reagent kits.

-Tom

jMHC has a GUI and does this well.

Works much faster on the mac, and with large data sets you will need to open the java script from the terminal, allocating more RAM

[monia]

Hi,
I have 96 samples and i want to design barcodes. How can't do it?
Thax for your help