Go Back   SEQanswers > Applications Forums > Metagenomics

Similar Threads
Thread Thread Starter Forum Replies Last Post
Distinguish sequencing errors and real mutations in mitochondria data cindylanzao Bioinformatics 4 07-20-2017 01:30 AM
Mantain mapping reads to chloroplast and mitochondrial wheat genome? DianaD RNA Sequencing 0 08-08-2016 10:08 AM
Reads amount - chloroplast genome assembly Ahaswer Bioinformatics 0 02-19-2014 12:17 AM
Distinguish real duplicates from those generated by PCR aquleaf Bioinformatics 1 10-30-2012 05:49 AM

Thread Tools
Old 11-19-2019, 01:20 AM   #1
Junior Member
Location: Italy

Join Date: Sep 2019
Posts: 5
Question How to distinguish between 16S chloroplast reads and real ones?


In an old experiment dating back to 2014, some 16S libraries from plant samples were prepared, with the aim to identify the microbial community living/contaminating the surface of the fruits.

The primer choice was the couple 27F and 533R.

Sadly, once analyzed with QIIME2 and clustered, out of 12 samples, 10 had chloroplast DNA contaminations percentages up to 99% (80-99%), making them completely useless.

I'd like to run the experiment again, with Illumina technology, addressing the problem of the primers not being specific enough to distinguish between plastid and bacteria DNA.

At the moment, the only scientific paper directly addressing the issue I was able to find was this one, but it's quite an old one.

Any suggestions here?
Thanks in advance.
Light92 is offline   Reply With Quote
Old 11-26-2019, 06:11 AM   #2
Junior Member
Location: New Brunswick, Canada

Join Date: Nov 2014
Posts: 3

I too am having the issue when looking for endophytes in leaf and stem tissue. Qiagen DNeasy as well as Qiagen Kingfisher soil kit extractions then using V4 region as well as V5/6 are not working well for me on our MiSeq. What are others doing to reduce chloroplast and yet have a good and reliable region to survey?
seanw is offline   Reply With Quote
Old 11-26-2019, 12:14 PM   #3
Location: Canada

Join Date: Jun 2013
Posts: 56

Have you tried or considered using a blocking oligo (such as one with a C3 spacer) designed to reduce amplification of the "host" plant tissue? A quick look at an alignment of nucleotide data might tell you whether there are any good candidate blocking oligo sites.
ATϟGC is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 09:56 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO