Hello,
I would like to ask you if you have any experience with multiplexing of 384 samples per run on Illumina NextSeq.
We are using SNP assay designed by Roche = SeqCap EZ Choice library with 3500 SNPs included.
What barcodes chemistry should we use for 384 samples? We found that Swift Biosciences is offering Combinatorial Dual Indexing primer kit (Cat.No. 489768) which should allow you to multiplex up to 768 samples. But is it feasible?
Thank you very much for your help and advice!
Tereza
you can order your own index primers (e.g; 100nM form companies like idtDNA tech.) if you you are going to do more runs in future
Dear Ahmad,
many thanks for your answer.
So, you think that it is possible to have 384 samples in one run with suitable coverage (=I mean at least 50x for each SNP).
It depends on the method you are going to use and more importantly the genome size of your organism. What organism you are working on? hypothetically it is possible (400 million reads for nextseq), but if your species has large genome it is safe to pool much lower number samples. Best is to contact to genomic facility btw: why do you need such high coverage?
Bests,
Farhad
Dear Ahmad,
we are sequencing only SNPs, using fragmented human DNA + targeted capture with 3000 capture probes. We have successfully tested them on 96 samples in one run but I am not sure if 384 samples will be also ok. I think that it should work as it is not whole genome nor exome sequencing but I am afraid of low coverage.
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