SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
low intensity signal with peaks under peaks mohd2b Sanger/Dye Terminator 5 11-19-2014 07:11 PM
Unwanted Small (~80bp) Bioanalyzer Peaks Daytwa Sample Prep / Library Generation 0 04-13-2013 06:00 PM
RNA quality - many peaks megalodon Sample Prep / Library Generation 1 02-25-2013 12:56 PM
SNP quality distribution peaks at 222 from variant call pile elfuser Bioinformatics 0 07-31-2012 11:42 AM
200bp kit with 316 chip + question: larger fragments selection Gorbenzer Ion Torrent 1 06-08-2012 09:13 AM

Reply
 
Thread Tools
Old 08-19-2015, 02:14 AM   #1
mzahir89
Junior Member
 
Location: Groningen, The Netherlands

Join Date: Aug 2015
Posts: 6
Post Tapestation quality peaks show unwanted larger fragments.

Hi all,

I used Covaris to shear human genomic DNA at a set size of 300bp and 200bp respectively. This is to sequence my samples using NEB library prep kit on a MiSeq platform.

I have however noticed, although both versions of Covaris programs set seem to work, I have also a rather significant portion of my DNA quality peak larger than 200 and 300bp as initially expected. The 200bp peak extends to 700bp whilst the 300bp extends to 1000bp. (File attached)

My questions are:
1. Is this ok to proceed for sequencing? How will it effect my overall coverage and sequence quality?
2. If i decide to fine tune my protocol for size selection, will i lose important sequence information?
3. I am not a big fan of Tagmentation using Transposase (which is why I prefer Covaris). However, say I use Tagmentation method to shear my DNA (and Nextera for Lib prep), will I see similar peaks? Is size fragments larger than desired ok for sequencing?
4. What can i do to avoid getting the extended fragment peaks? In other words, how do i narrow the size of the peaks (without size selecting them)

Any advice and help will be deeply appreciated. Thanks in advance
Attached Files
File Type: pdf D1000 Report_TLA_Covaris_Test.pdf (223.5 KB, 44 views)

Last edited by mzahir89; 08-19-2015 at 02:20 AM.
mzahir89 is offline   Reply With Quote
Old 08-19-2015, 04:22 AM   #2
nucacidhunter
Senior Member
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,171
Default

1- If they are your final libraries, a left side bead cut would help to remove fragments with small inserts. If they are sheared DNA you can do the left side cut prior to library prep step. Larger fragments will not affect sequencing if libraries are quantified correctly by qPCR.
2- No, but library diversity (# of unique reads) is depend on input amount among other factors. Whether size select or not depend on availability of DNA and downstream application of data.
3- Nextera library size distribution range will be wider than your sheared DNA.
4- DNA can be sheared to shorter fragments which will result in narrower size distribution but then you need to adjust number of sequencing cycles to library’s average insert size.
nucacidhunter is offline   Reply With Quote
Reply

Tags
covaris, fragment analysis, fragment size, nextera, tapestation

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 01:10 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO