16S sequencing tissue - host DNA contamination

KateShepard · 06-23-2017, 02:04 AM

Dear all,

We are planning to use human/mouse intestinal tissues/biopsies for V4 microbiome sequencing. I am worried that the large amount of host DNA from the tissue will result in amplification of host mitochondrial DNA.

Does anyone have experience of V4 sequencing of tissue samples or biopsies, and if so is there usually a high proportion of mitochondrial sequences present? Are there any strategies to reduce host mitochondrial amplification?

Thanks for your advice,
Kate

GenoMax · 06-23-2017, 04:28 AM

Someone else will comment on experimental tricks to mitigate this issue but I wanted to mention BBSplit from BBMap suite which is designed for exactly this bioinformatics task. You can find the thread for BBSplit here.

thermophile · 06-23-2017, 11:47 AM

The 515F/806R primers that are out there (Caparosso or Kozich primers) are designed to be biased against chloroplast and mitochondria. My tricks for tissues are use a ton of DNA because most of what you are measuring is host, also spike in a tiny amount of non-illumina/non-barcoded 515f/806r

adam.geber · 08-15-2017, 10:00 AM

Hi thermophile,

Can you clarify the purpose of spiking in the non-barcoded primers to minimize host mitochondrial contamination? Wouldn't you expect these to bind equally well to the bacterial V4 region?

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06-23-2017, 02:04 AM	#1
KateShepard Junior Member Location: UK Join Date: Apr 2016 Posts: 5	16S sequencing tissue - host DNA contamination Dear all, We are planning to use human/mouse intestinal tissues/biopsies for V4 microbiome sequencing. I am worried that the large amount of host DNA from the tissue will result in amplification of host mitochondrial DNA. Does anyone have experience of V4 sequencing of tissue samples or biopsies, and if so is there usually a high proportion of mitochondrial sequences present? Are there any strategies to reduce host mitochondrial amplification? Thanks for your advice, Kate

06-23-2017, 11:47 AM	#3
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	The 515F/806R primers that are out there (Caparosso or Kozich primers) are designed to be biased against chloroplast and mitochondria. My tricks for tissues are use a ton of DNA because most of what you are measuring is host, also spike in a tiny amount of non-illumina/non-barcoded 515f/806r __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

06-23-2017, 04:28 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,091	Someone else will comment on experimental tricks to mitigate this issue but I wanted to mention BBSplit from BBMap suite which is designed for exactly this bioinformatics task. You can find the thread for BBSplit here.

08-15-2017, 10:00 AM	#4
adam.geber Member Location: United States Join Date: May 2014 Posts: 40	Hi thermophile, Can you clarify the purpose of spiking in the non-barcoded primers to minimize host mitochondrial contamination? Wouldn't you expect these to bind equally well to the bacterial V4 region?