Low DNA concentration and Illumnia MIseq

[mumairba]

Hello all,

I want to do Illumina miseq on total DNA extracted from feacal microbes to check the relative abundance of different microflora.

Problem is that i am getting very low DNA concentration the samples i am using to extract DNA, I am using Qiagen stool DNA extaction kit and the concentration i am getting on Nanodrop 2000 is very low ( I.e. less than 10 ng/ul in most of the sample).

I have also performed gel electrophresis to check the molecular band of my total DNA, and the band i am getting is very shallow against Hind II Lamda marker.

I want to know if that concentration is enough to do PCR amplification for the Illumina Miseq to get the sequencing library ?

Regards,

Umair

[luc]

Hi Umair,

this amount of DNA can be sufficient.
You want to sequence the genomic DNA sequences and not 16S sequencing?
There are plenty of low input genomic library prep protocols around: Qiaseq UltraLow Input, Rubicon Genomics Thruplex, Kapa Hyper, NEB Ultra, BiooScientific,
For metagenomic sequencing usually PE150 bp reads on HiSeq sequencers are used.

[mumairba]

Sorry if i didn't make it clear in the first post, i am new to next gen sequencing.

Usually for gut microbes we do 16s sequencing, so i will do 16s sequencing...

Do you think that this concentration is enough for 16s ?

[kmcarr]

Sorry if i didn't make it clear in the first post, i am new to next gen sequencing.

Usually for gut microbes we do 16s sequencing, so i will do 16s sequencing...

Do you think that this concentration is enough for 16s ?

Your DNA is more than adequate for 16S metagenomics. 16S libraries are created entirely by PCR so only a tiny amount of input DNA is required. 16S amplicon sequencing is nearly always done with PE250 reads on the MiSeq.