Tapestation quality peaks show unwanted larger fragments.

[mzahir89]

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

[nucacidhunter]

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.