SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
How to pool ATAC-seq libraries in equimolar concentrations? ECorrales Sample Prep / Library Generation 4 11-22-2019 02:39 PM
Target 16S V4 rRNA and ITS genes in one primer pool PerkinElmer AG Vendor Forum 0 03-11-2019 06:58 AM
Adapter artifacts 16S library prep from low density samples desertwillow Sample Prep / Library Generation 1 05-22-2014 02:43 AM
stuck in normalize and pool step TruSeq anamar Sample Prep / Library Generation 0 03-01-2013 08:18 AM
How to normalize the bam files from different samples ykdang Bioinformatics 0 11-07-2012 12:12 PM

Reply
 
Thread Tools
Old 03-21-2020, 09:09 AM   #1
samd
Member
 
Location: California

Join Date: Nov 2019
Posts: 14
Default How to normalize/pool a 16S library when samples are highly variable concentrations??

Hi all,

I've been having this issue that has been plaguing my PhD. Some of my sequencing runs come back with terrible uneven read coverage. I am guessing it is a pooling problem but still not 100% sure.

For some background on my pipeline: after my final bead clean step I quantify all my samples and usually get a huge range of concentrations from 5ng/ul to 100+ ng/ul. This isn't surprising as my gut microbiome samples come from a variety of fish species. I then proceed to pool the samples by adding different volumes to create a 20ng/ul final pooled library. Theoretically, I am adding the same amount of moles of each sample to the pool. Obviously it isn't perfect but I do not understand why my read coverage is so terrible. I usually get back many samples with <1000 reads and some with over 300k reads on an Ilumina Miseq (16S V4 amplicon).

My question is: is there a different way to normalize the concentrations before pooling?

Is diluting samples with water to normalize concentrations (adding different amount of H20 to get them all the same concentration) helpful before pooling?

Are highly variable sample concentrations prior to pooling a culprit for uneven read coverage?

For indexing (2nd PCR), I usually add 1ul of PCR product or 2ul depending on the brightness from the gel. This has worked fine before but would it help if I quantified my PCR products and then added a more precise volume for indexing to keep it more normalized?

I pool in a way so I am adding a minimum of 2ul so as to avoid pipette error with small volumes.

Some additional info:
I do a 2-step PCR process with 515-806R primers
Qiagen multiplex kit for PCR 1 and Kappa Hi-Fi for PCR 2
Nextera Unique Dual indexes for indexing
Omega RXN Pure Beads for bead cleans
Qubit for quantification

Any help would much be appreciated!!
Cheers,
Sam

Last edited by samd; 03-21-2020 at 09:36 AM.
samd is offline   Reply With Quote
Old 03-22-2020, 02:45 AM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,231
Default

High variability of concentration could be one reason.

To reduce variability:
1- Quantify 1st PCR amplicons and use the same mass for indexing PCR. This will reduce gel invisible artefacts.

2- Normalize 2nd PCR in two steps, first to 10 ng/ul and then to 5 ng/ul. I think normalizing to 20 ng/ul is too high.

3- If PCR yield is not limiting use 3 ul for Qubit quantification

You still will see variability but it should be limited max 2x. This will increase hands on time but depending on your workload might worth.
nucacidhunter is offline   Reply With Quote
Old 03-23-2020, 06:41 AM   #3
samd
Member
 
Location: California

Join Date: Nov 2019
Posts: 14
Default

@nucacidhunter Thank you for this detailed answer. All these make sense especially the 2 time normalization. Why is lower concentration better? More accurate?
I will employ these modifications once things return back to normal.
Thanks again.
samd is offline   Reply With Quote
Old 03-26-2020, 05:53 AM   #4
MU Core
Member
 
Location: Columbia, Missouri

Join Date: Apr 2008
Posts: 57
Default

Do you normalize your template input for the 1st PCR? We have found this very useful and critical in getting an even representation of reads in the pool. As long as there are no PCR inhibitors in the sample (i.e. similar PCR efficiency in each reaction), we are able to pool all wells across a 96-well plate by equal volume and perform a single bead clean-up. The variation in read count across samples is minimal.
MU Core is offline   Reply With Quote
Old Yesterday, 08:56 AM   #5
samd
Member
 
Location: California

Join Date: Nov 2019
Posts: 14
Default

@MU Core I actually also have gotten similar advice from someone at a sequencing core. The one thing is that I do have huge variability in PCR efficiency and output. However I feel like this pre-normalization step combined with what @nucacidhunter said would greatly help my problem.

As for the beadclean step, are you saying that you simply do a single beadclean on the pooled library? That would save me a great amount of time.
Cheers,
Sam
samd is offline   Reply With Quote
Old Yesterday, 10:23 AM   #6
MU Core
Member
 
Location: Columbia, Missouri

Join Date: Apr 2008
Posts: 57
Default

That's correct, a single bead clean-up on the pool. As long as you have good amplification across the plate and normalize template input amount, there is no need to quantitate and normalize the PCR products.
MU Core is offline   Reply With Quote
Reply

Tags
16s, library prep, microbiome

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 04:57 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO