16s MiSeq Illumina library concentration and PhiX

[BioGenomics]

I had another question concerning low cleaned amplicon concentration before indexing PCR. Can the Nextera indexing PCR cycle (8 cycles) be increased (to 10-12) without any issues to increase the concentration before final bead clean-up ? Also, what's the contamination likelihood using a SpeedVac ?

cheers

[nucacidhunter]

Illumina’s guide for clustering libraries on MiSeq can be find by google searching “Preparing Libraries for Sequencing on the MiSeq”. 4 pM is the concentration for denaturation step. Final pM concentration for clustering varies among labs as the quantification systems and practices differ. For amplicons less input improves PF% and also quality scores which usually is achieved with cluster densities below 1k. Illumina also recommends incubation of HT1 diluted denatured libraries at 96°C for 2 min before loading to improve clustering GC rich fragments

[BioGenomics]

Thanks for this nucacidhunter. So is 4 pM the optimal (clustering) conc for amplicon Seq ?

cheers

[nucacidhunter]

Anyone having bad (low) clustering doing this or no problem when using the v3 MisEq kit (2 X 300 bp) ?

We saw inconsistent cluster density. Illumina recommends concentration by SpeedyVac. Other options would be concentrating them using column or AMPure beads by reducing elution volume.

The protocol says 5 % (v3 kits again), but the rep says 10-20 %. Any comments on this ?

Illumina's current official recommendation with the latest software is 5% PhiX spike-in, but 10% spike-in increases the likelihood of successful run.

[BioGenomics]

Hi all,

I have 2 small questions concerning the 16s sequencing protocol for MiSEq:

1) Some samples have very low concentration so we are considering loading a 2pM library instead of the more standard 4pM. Anyone having bad (low) clustering doing this or no problem when using the v3 MisEq kit (2 X 300 bp) ?

2) There are various older threads discussing the PHIX % to be added to the library. The protocol says 5 % (v3 kits again), but the rep says 10-20 %. Any comments on this ?

thanks

Greg