SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Sequence in (biological) duplicate or not? Jasseq RNA Sequencing 3 09-15-2015 05:37 AM
Sequence of PhiX MiSeq primers Oregano Illumina/Solexa 4 09-15-2014 03:46 PM
When is a gene considered unexpressed? lukas1848 Bioinformatics 2 11-07-2013 01:19 AM
Sorting fastq by primers, then searching by sequence (with mismatches) jme Bioinformatics 0 01-18-2012 09:25 AM
Important fields of SAM file to be considered while compression nadir Bioinformatics 0 03-29-2011 09:58 PM

Reply
 
Thread Tools
Old 04-01-2019, 09:45 AM   #1
sformel
Junior Member
 
Location: New Orleans, LA

Join Date: Apr 2019
Posts: 4
Default should primers be considered biological sequence?

Hi,

Forgive me if this is naive, but I'm still newish to thinking about sequencing. I've begun working with the DADA2 software and the tutorial clearly states that primers should be trimmed because they are non-biological nucleotides.

I'm not arguing against it, but I don't fully understand it. If the primer matches a sequence of DNA, then shouldn't it be just as legitimate a sequence as the sequence derived from the sequencing process?

Any thoughts?

Thanks,
Steve
sformel is offline   Reply With Quote
Old 04-01-2019, 09:49 AM   #2
jhalpin
Member
 
Location: Atlanta, GA

Join Date: Jan 2015
Posts: 25
Default

Primers and adapters are added to the fragments during the preparation of the samples. They are not native sequences and are extremely over represented compared to any natural occurrences of those short sequences.
jhalpin is offline   Reply With Quote
Old 04-01-2019, 10:04 AM   #3
sformel
Junior Member
 
Location: New Orleans, LA

Join Date: Apr 2019
Posts: 4
Default

Thanks for the quick reply! But I'm still not sure I understand.

My thought is that the because the primer matched the DNA fragment during PCR, it can be considered a legitimate copy of a biological sequence of nucleotides, just like the copy that is made during sequencing.

But it seems from what you wrote that the problem is due to over representation of those sequences. How is a primer sequence different from a highly conserved region of an amplicon?

To be clear, I'm coming at this from a metagenomics perspective, which may be skewing my thoughts.

Last edited by sformel; 04-01-2019 at 10:06 AM. Reason: picture was too large
sformel is offline   Reply With Quote
Old 04-01-2019, 10:41 AM   #4
jhalpin
Member
 
Location: Atlanta, GA

Join Date: Jan 2015
Posts: 25
Default

Sorry.. in the lab to make sequencing libraries the primers are added. They aren't in the sequence of the thing you're looking at. The primers are constructed by a company and sold as part of the kit. Here's an overview of the lab side: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351865/
jhalpin is offline   Reply With Quote
Old 04-01-2019, 11:12 AM   #5
sformel
Junior Member
 
Location: New Orleans, LA

Join Date: Apr 2019
Posts: 4
Default

Ok, I understand what you're saying in regards to RNA-seq type libraries.

However, I'm working with a 2-step PCR in which the first step is an amplification of a marker gene, followed by an indexing PCR (addition of adapters and barcodes). So in my case, the primer is part of my biological sequence.

In any case, I appreciate your thoughts. I'm going to think on it a little more and see if it clicks.
sformel is offline   Reply With Quote
Old 04-01-2019, 11:22 AM   #6
cmbetts
Senior Member
 
Location: Bay Area

Join Date: Jun 2012
Posts: 102
Default

That sequence isn't derived from the fragment you're trying to sequence. 100% of it was chemically synthesized by your oligo synthesis company. It match close enough to your insert of interest to prime it, but does not guarantee that the sequences were perfectly complementary. For instance, any mispriming artifacts will perfectly match your sequence of interest over the length of the primer...
cmbetts is offline   Reply With Quote
Old 04-01-2019, 11:34 AM   #7
sformel
Junior Member
 
Location: New Orleans, LA

Join Date: Apr 2019
Posts: 4
Default

Ah ok, that makes sense. I didn't think about those kind of biases. Thanks for your help!
sformel is offline   Reply With Quote
Old 04-02-2019, 05:07 AM   #8
ATϟGC
Member
 
Location: Canada

Join Date: Jun 2013
Posts: 45
Default

I will just echo cmbetts in saying that you should always remove your oligo/primer sequences from Sanger and High-throughput sequencing data since they are artificial. You can sometimes find primer/oligo sequences in "cleaned" sequences archived in GenBank and other public databases but this is not a good practice.

The only good reason I can think of off-hand to not trim the oligos is if you are archiving the raw data, in which case you should state the oligos used so others can trim them for downstream analyses.
ATϟGC is offline   Reply With Quote
Reply

Tags
dada2, primers

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 06:05 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO