How many samples can you load in one run using an Illumina MiSeq reagent V3 kit?

I'm trying to sequence amplicons of the 16S V3-V4 region (aprox. 540 bp). I've read that at least you need 10,000 reads per sample to characterize a microbial community. If I'm expecting at least 20,000 reads per sample, how can I know how many samples can I sequence at the same time? Aside from the limit of barcodes. We are planning to use a 2 x 300 bp run. Thank you in advance.

I am new in the world of NGS Platforms. I am interested mainly in RNA-seq with illumina ( 100+bp paired end reads) and I dont Know how much throughput I am going to get if I mix 15 samples in one lane?

Hello researchers,

I am doing a dual-indexed 16S amplicon microbiome characterization of salmon gut using MiSeq (250bp; paired-end). I have a total of 180 samples from which I need to achieve more than 10K reads per sample for the alpha and beta diversity analysis. During my past runs where I had ran 150 samples in one run, I ended up with less than 10K for most of the samples.

My question is should I divide my 180 samples and sequence in two different runs?

(I think, running less samples could give a better sequencing depth.)

Thank you.

I am trying to make a DNA library for the Illumina NGS. The kit I am using for is NEBNext Ultra DNA library Prep kit for Illumina. The starting material is the cDNA. So far this kit works fine for me. But when I trried to clean up the PCR pruduct with Ampure XP beads in last step. I lost most of DNA. For example, I started with 400ng cDNA as the material. After 1st clean up with beads, I got 90% of DNA for the following PCR step, the 260/280=1.99, 260/230=3.4. Then I got about total 15ug of PCR product, concen is 300ng/ul, 260/280=1.75, 260/230=0.88. But when I applied the last step of clean for those PCR product, the nanopdrop showed wield data like -2 or 0.1 ng/ul, and 260/280= 3, 260/230=1. I tried so many times, communicated with the tech support, but totally didn't work. The gel test showed nothing. 

I am confused, before PCR, I used beads to clean up DNA, every thing works ok. But after PCR, when I used the same procedure and beads to clean up the PCR product, I lost all PCR product. Totally frustrated. My back up plan is sending out the library without the last step clean up. Hopefully that will be fine. Anybody got any suggestion for that? I will appreciate that so much.  

We collected faecal samples from various species that we want to analyse their microbial communities' DNA via Next-Generation Sequencing. The problem is that due to budgetary constraints, we will have to pool the isolated DNA from each sample to be able to analyse them all. We will of course keep each species separated from the other ones, and we are planning to pool the samples from individuals which share enclosure only. Nevertheless, we do not know if there is a maximum number of samples that can be pooled together for NGS.

The library was QCed with average size around 772. The NaOH is prepared freshly from 1N stock to 0.2N. I could not recall any mistake in the process. However, my miseq run gives me cluster density 670K/mm2 of chemistry v2 300 and 0%PF. I spiked in 1%Phix. Does anyone have comment on this?

Hi,

I'm just curious if I am able to obtain good quality data of 600 bp read length using MiSeq v3 600 cycle kit.

I know the read quality goes down with longer read length.

Has anyone tried using this kit to get longer read lengths?

Other question is, is there any good NGS technique / platform that yields long read lengths (~500 bp>) ?

Thanks for all the help

what is the role of the index primers in the illumina sequencing and how they differentiate the reads?

How many reads/sample is required for species level identification when performing 16S rRNA sequencing of the gut microbiome?