Microbial Metagenomics: Nextera. Nextera XT, or TrueSeq to run on HiSeq 2500?

[robotbanni]

We have 96 gut microbiome samples that we want to run using the Nextera XT or Nextera Kit. Most of our samples have between 1 - 40 ng of DNA. Would it be best to dilute our samples, we dont know how that would affect library quality. We want to run it on the HiSeq 2500 but a previous thread said that the bead normalizing step would be skipped and qPCR was recommended by Illumina (which is best for us since we are limited by budget and we would have to purchase the Amp XP beads and a magnetic plate). Or would it be better to use the TrueSeq kit? (first time doing library prep)

Best,
Maria

[nucacidhunter]

I would consider following:
1- Available DNA ( 1 ng for Nextera XT, 50 ng for Nextera and 100 ng for TruSeq)

2- Complexity or size of genome: in this case it would be sum of all microbiome genome. If the expected microbiome species is high then Nextera XT will not provide sufficient complexity.

3- Available DNA is not sufficient for either of two remaining kits.

You will need a kit that can prep highly diverse libraries with lower inputs. In any case cost of library prep will exceed sequencing cost (if you are planning one lane of sequencing).

We want to run it on the HiSeq 2500 but a previous thread said that the bead normalizing step would be skipped and qPCR was recommended by Illumina (which is best for us since we are limited by budget and we would have to purchase the Amp XP beads and a magnetic plate). Or would it be better to use the TrueSeq kit? (first time doing library prep)

I think that thread about bead normalisation refers to Nextera XT workflow which is supplied with kit. You will need Amp XP and magnetic stand regardless of library prep method because all will have a bead clean up step.

[jlei_face]

What is the end goal of this? Taxonomic assignment or de novo reconstruction of the samples? How many samples do you need to sequence?
Additionally, I would recommend skipping the bead normalization (as you said) and just manually normalize manually, and while qPCR will give you accurate quants, you can also use a fluorometric method (like a picogreen assay) which is much cheaper and faster.

[PGT_Bordeaux]

In my facility, we use a 16S PCR with adaptator illumina in 5' primer. After this step we purify with XP AMpure (Beckman) or Clean NA (MagBio-mokascience). For quantification we use high sensitivity chip on bioanalyzer and manually normalize.
For sequencing, I think MiSeq is better because you will need a good lenght for taxonomic identification (2x 250 or 2 x 300).

16S PCR (V3, V4 or both) will provide you a high multiplexing rate instead if you read all whole genome in your samples.

best,