Go Back   SEQanswers > Applications Forums > RNA Sequencing

Similar Threads
Thread Thread Starter Forum Replies Last Post
NGS result-laboratory report disclaimer A-R-T Clinical Sequencing 0 01-10-2015 10:27 PM
How to translate RNA-Seq result to normalized gene expression level in qRT-PCR? ymc RNA Sequencing 0 12-18-2013 03:32 PM

Thread Tools
Old 11-24-2020, 11:18 AM   #1
Junior Member
Location: Charlottetown, PE, Canada

Join Date: Apr 2017
Posts: 4
Default NGS result that can't be verified with RT-PCR

I received 5 plant leaf samples for NGS. Two of the 5 were determined to be positive for virus via RT-PCR. However when I performed NGS, all 5 samples were determined to be positive. In the 3 samples that were supposed to be negative, I acquired complete genomes when assembled using CLC Genomics Workbench.

I can't seem to find any source of contamination. The 3 supposed negative samples are of a different strain than the other two. However, it is a strain that should still be picked up by my RT-PCR primers.

I hadn't used these indices in a while as I know that I have had problems with false positives in the past when using the same index sequences in consecutive runs. This is why I always rotate my index sequences for each NGS run. The last several times these index sequences were used, they did not detect that target. So, I'm thinking that it's not that type of contamination.

I've ruled out that it's pipette contamination because I'm using the same pipettes on library prep as I do with RT-PCR.

The run was a near perfect 1452Kmm-2. So, overclustering leading to strange base calls can also be ruled out.

Does anyone have any ideas what could be going on here?
dlhmmll is offline   Reply With Quote

false positives, rna-seq, rt-pcr

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 09:29 AM.

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