Go Back   SEQanswers > Applications Forums > RNA Sequencing

Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-seq technical replicates at library construction level obig Bioinformatics 9 08-16-2012 12:43 AM
Need a paired-end RNA-Seq library construction protocol satp Illumina/Solexa 1 08-11-2012 07:39 PM
total rna quality for library construction wayland Illumina/Solexa 3 10-13-2011 06:10 AM
total RNA quality for library construction neveaire Sample Prep / Library Generation 4 04-05-2011 12:39 PM

Thread Tools
Old 11-08-2017, 07:23 AM   #1
Location: U.S.

Join Date: May 2017
Posts: 21
Smile strong 140bp peak following RNA library construction

Hello all,
I have created an illumina truseq library on bacteria by first performing ribozero rRNA depletion kit and then performing a truseq stranded mRNA prep while skipping the poly-A selection reaction. my libraries are complete and in cDNA format. i have run a DNA 1000 bioanalyzer assay to visualize the libraries. we see the desired ~260 bp library peak but in every sample there is also a strong ~140bp sharp peak which I think will throw off my molarity calculations (what i'm actually thinking about is proper flow cell cluster density), if it is say, a product of the library prep which cannot form clusters. in certain circumstances it seems to be in much higher concentration than the fragments that i want to sequence. does anyone know what this ~140bp sharp peak is in every sample and i'm also interested to know if there is some way of accounting for this when i do my normalization dilutions pre-pooling. thank you
Attached Images
File Type: gif 20171108.gif (121.0 KB, 35 views)
seqtechno1 is offline   Reply With Quote
Old 11-08-2017, 07:28 AM   #2
Senior Member
Location: Bethesda MD

Join Date: Oct 2009
Posts: 509

The band is adaptor dimers. They form clusters. Use gel or bead purification to remove.
HESmith is offline   Reply With Quote
Old 11-08-2017, 07:36 AM   #3
Location: U.S.

Join Date: May 2017
Posts: 21

Hi HESmith. thanks for your input. the library construction finishes with two separate ampure xp bead purifications. do you think it is worth performing again?
seqtechno1 is offline   Reply With Quote
Old 11-08-2017, 08:09 AM   #4
Senior Member
Location: New England

Join Date: Jun 2012
Posts: 200

You don't need to perform the bead cleanup/size selection to remove the larger fragments, just the smaller fragments, but you do need to remove them because they will take over a large portion of your sequencing run. You really shouldn't have ended up with so much adapter dimer in the first place - be very careful about adding the correct amount of beads.
microgirl123 is offline   Reply With Quote
Old 11-09-2017, 02:16 AM   #5
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238

It seems that duel index adapters has been used and clean up has been inefficient in removing excess adapters. The best way will be to tackle it after adapter ligation by reducing clean up bead ratio.

These libraries can be cleaned up one more time. To avoid over clustering due to adapter-dimers you need to use a region covering adapter-dimers as well for average size calculation.
nucacidhunter is offline   Reply With Quote
Old 11-12-2017, 04:55 PM   #6
Location: KiwiTeritory

Join Date: Sep 2014
Posts: 19

I wonder what brand of beads you have used for clean ups and if they have been used according to TruSeq protocol.
BioKiwi is offline   Reply With Quote

libary prep, quantitation, ribo-zero, rnaseq, truseq stranded mrna

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 09:10 AM.

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