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Old 08-24-2017, 10:06 PM   #1
nvd
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Default Miseq multiplexed sequencing with NEB library prep kits

Hi all,
I am new to deep sequencing and wish to receive your advice.
I am going to use NEBNext Ultra Directional RNA Library Prep kit with NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1) and Miseq reagent kit v2.
I have several questions below
1) The index primer set 1 include a PCR universal primer and 12 index primers. I suppose dual-indexed library prep is impossible with this index primer set, am I right?
2) If so, I can only perform multiplexed single-read sequencing, NOT multiplexed paired-end sequencing as the latter requires dual-indexed libraries?
3) If multiplexed single-read sequencing is the only possibility, I should prepare libraries longer than 300bp and run 300 cycles?
Thank you very much in advance.
nvd
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Old 08-24-2017, 11:12 PM   #2
nucacidhunter
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You can do paired end multiplex sequencing for libraries prepared with single indexed primers.
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Old 08-25-2017, 10:57 AM   #3
nvd
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Hi nucacidhunter,
Thanks for your prompt response. Do you mean at the PCR enrichment of adaptor ligated DNA, I use only 1 index primer/sample and no universal PCR primer? Then the index primer will be at the two ends of the library?
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Old 08-25-2017, 11:19 AM   #4
nucacidhunter
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You need to use one index primer and the universal one otherwise PCR will fail. In Illumina sequencing index read is seperate from R1 and R2. It identifies the strand in a cluster that can be sequenced as single (one end) or paired end.
You will need also pay attention to index compatibility if multiplexing less than six libraries.
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Old 08-25-2017, 12:28 PM   #5
nvd
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Many thanks for your clarifications. I got you and understand why indexes are read separately from R1 and R2 now. I greatly appreciate your useful advice.
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