Illumina
Nextera XT Indext Kit
FC-131-1001
I use Nextera XT Indexing (24 indices) kit from Illumina to prepare whole genome sequencing libraries for bacterial isolates. Each run for MiSeq platform, I have successfully multiplexed my samples up to 64 using this indexing kit without any problem. Quality and separation of reads post sequencing were very good enough to do comparative sequence analysis.
Whole genome sequencing in Illumina MiSeq platform
Genomic DNA from bacterial isolates from soil samples
Tagment the genomic DNA and then proceed for PCR amplification of libraries using Nextera Index Kit. Use i7 and i5 primers in the kit for multiplexing the samples as per your need and number samples to be multiplexed. Use Illumina Truseq Index plate fixture to aid in adding indexing primers to the samples (as given in the instruction manual). Perform required number PCR cycles (which very depending on input amount of genomic DNA). Clean up post PCR libraries and then normalise and pool libraries. Then proceed for sequencing.
Recommend using TruSeq index plate fixture to aid in adding indexing primers without confusion.
Successfully multiplexed samples up to 64. Post sequence qualities for all 64 samples were good enough to perform comparative sequence analysis.
N/A
Easy to follow and less hands on time during experimental steps.
None
Recommended for automatic (robotic; in 96 well format) high throughput amplicon sequencing.