SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Homemade AMPURE beads size selection issue Todd McLay Sample Prep / Library Generation 0 06-19-2014 07:23 PM
ddRAD size selection with Ampure beads Todd McLay Sample Prep / Library Generation 3 05-06-2014 08:31 PM
miRNA library size selection using Ampure beads aparnanatarajan Sample Prep / Library Generation 0 07-25-2013 01:49 PM
AMPure XP Beads Size Selection Chemistry ALBraun Illumina/Solexa 3 02-22-2012 11:06 AM
double AMPure size selection Bjorn Sample Prep / Library Generation 0 10-09-2010 02:36 PM

Reply
 
Thread Tools
Old 01-17-2017, 12:53 AM   #1
Justin87
Junior Member
 
Location: Cape Town, South Africa

Join Date: Jan 2017
Posts: 4
Default Double Size Selection (500 bp) with AMPure XP Beads

Good day,

Could somebody please elaborate on the ratios of Ampure XP beads required for DOUBLE-SIZE SELECTION to achieve an average fragment size of ~500 bp.

I had followed an illumina protocol: Improved Protocols for Illumina Sequencing (Alternate Protocol 2, Ampure Bead Double DNA Size Selection; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849550/).

Briefly, an initial 0.65X addition of reagent:sample was followed by discarding the beads (bound to larger unwanted fragments) and transferring the supernatant to a clean tube. I had then added a 0.12x ratio of reagent to (input) sample volume. The supernatant was then discarded, and the DNA eluted from the beads. However, based on Bio analyzer results, the trace dips at the ~500 bp section but rather produces TWO peaks on either side...

Please help!!!

Last edited by Justin87; 01-17-2017 at 12:55 AM.
Justin87 is offline   Reply With Quote
Old 01-17-2017, 01:36 AM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 977
Default

Possible reasons:
1- bad Bioanalyzer run
2- starting material size distribution
3- skipping Ethanol wash
nucacidhunter is offline   Reply With Quote
Old 01-17-2017, 01:55 AM   #3
Justin87
Junior Member
 
Location: Cape Town, South Africa

Join Date: Jan 2017
Posts: 4
Talking

Thanks for your response, nucacidhunter!

I repeated the bio-analyzer run twice, all with very similar results, so that rules out a potentially bad run.

We are pursuing a dd-RAD sequencing experiment. So to reduce complexity of the plant genome, two of the same DNA samples had been digested separately with different combinations of restriction enzymes. This had produced a great smear of products for both as seen on the agarose gel. I also ran these digested products (before size selection) on the bioanalyzer chip, and it displayed a good range of fragmentation, even though the profiles between the two different enzyme combinations were slightly different.

I had also washed the final beads twice with freshly prepared 80% EtOH...

With regards to the double digestion protocol, is the ratio of the SECOND round (0.12x) of bead reagent calculated according to the INPUT volume used in the beginning of the experiment (50 uL, starting), or to the supernatant that was transferred after the FIRST round (0.65x) of bead binding (which would be 82.5 uL). I have read protocols with conflicting instructions...
Justin87 is offline   Reply With Quote
Old 01-17-2017, 05:53 AM   #4
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 188
Default

can you give volumes used throughout? I'm having a hard time figuring out what you did based on these ratios-.12x seems really small.
slide 62 demonstrates bead size selection pretty well. https://www.broadinstitute.org/files...PrepSlides.pdf
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 01-17-2017, 06:52 AM   #5
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 52
Default

It would also be helpful if you posted before and after traces from your size selection.
__________________
Josh Kinman
jdk787 is offline   Reply With Quote
Old 01-17-2017, 07:04 AM   #6
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 188
Default

Can you post a picture of your Bioanalyzer results? It sounds a bit like it might be overloaded - that can create a split peak.
microgirl123 is offline   Reply With Quote
Old 01-17-2017, 02:47 PM   #7
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 977
Default

Quote:
Originally Posted by Justin87 View Post
Thanks for your response, nucacidhunter!

I repeated the bio-analyzer run twice, all with very similar results, so that rules out a potentially bad run.

We are pursuing a dd-RAD sequencing experiment. So to reduce complexity of the plant genome, two of the same DNA samples had been digested separately with different combinations of restriction enzymes. This had produced a great smear of products for both as seen on the agarose gel. I also ran these digested products (before size selection) on the bioanalyzer chip, and it displayed a good range of fragmentation, even though the profiles between the two different enzyme combinations were slightly different.

I had also washed the final beads twice with freshly prepared 80% EtOH...

With regards to the double digestion protocol, is the ratio of the SECOND round (0.12x) of bead reagent calculated according to the INPUT volume used in the beginning of the experiment (50 uL, starting), or to the supernatant that was transferred after the FIRST round (0.65x) of bead binding (which would be 82.5 uL). I have read protocols with conflicting instructions...
It is usually to the starting volume. So if sample volume was 50 ul adding 30 ul bead will make ratio 0.6X. Adding 10 ul of bead to the supernatant will make the ratio 0.8X (50 ul sample, 40 ul bead in total).
I assume that you double digested DNA, checked on gel and Bioanalyser, size selected, ligated adapters, cleaned up and followed to PCR amplification and then run the PCR product on the Bioanalyser. If this is the case, then double peak could be result of amplification of repeat region and not an issue with size selection. In ddRAD only fragments in the selected size window and flanked by both restriction sites are amplified that could vary from sample to sample or digest to digest. Majority of digested fragments will be flanked by frequent cutter and will not be amplified so the profile of amplified fragments could vary significantly from that of digest.
nucacidhunter is offline   Reply With Quote
Old 01-17-2017, 11:25 PM   #8
Justin87
Junior Member
 
Location: Cape Town, South Africa

Join Date: Jan 2017
Posts: 4
Default

Hi All,

Appreciate all the feedback!

The DNA had been double-digested. We are performing size selection for an average fragment size of ~500 bp PRIOR to library preparation. Again, I had primarily followed a protocol published in a paper (link in my first post) trying to select for a similar-sized fragment. I had compared this to a few others, and the approach seemed quite logical. The exact approach/volumes used were as follows:

1) The input volume for the purification/size-selection was 50 uL.

2) A 0.65x ratio (32.5 uL) of AMPure XP Bead reagent was added. Placed on magnetic stand.

3) Supernatant (containing desired fragments) of ~82 uL was transferred to a new tube while the beads (bound to large unwanted fragments) were discarded (Bioanalyzer trace 2).

4) A 0.12x ratio (6 uL, based on the 50 uL input) of Ampure XP Bead reagent was added to supernatant from the previous step (I guess this results in a final ratio of 0.77x?). Placed on a magnetic stand.

5) The supernatant (containing the smaller unwanted fragments) was discarded (Bioanalyzer trace 3) while the beads (bound to the desired fragments) had been washed 2x with 80% EtOH and DNA eluted from the beads (Bioanalyzer trace 4).


I have attached the Bioanalyzer traces (High Sensitivity Chip) of:

1) the unpurified double-digested DNA (1:10 dil.)

2) the DNA eluted from the beads after the 0.65x AMPure XP addition (removing the larger fragments; 1:10 dil.). This is normally discarded.

3) the supernatant after the 0.12x AMPure XP bead addition (removing the smaller fragments) - this is normally discarded. The trace for this had consistently produced a strange profile where markers were not detected - based on my limited research, this is most likely attributed to the high salt conc.?

4) the DNA eluted from the beads after the 0.12x AMPure XP addition. This should contain the desired fragments, but instead produce two prominent peaks flanking the 500 bp region.


Oddly enough, an experiment I had worked on a few months back had produced a very similar situation (two peaks flanking the desired fragment region). When I analyzed the supernatant (after the second size cut), which is normally discarded, I had detected the single desired peak I was seeking.

Based on this, I am planning to clean up the 0.12x eluted supernatant from the experiment in question and visualize it on the bioanalyzer.

Apologies for all the repetition. I am relatively new to the NGS world, so thank you all for the patience.

Regards,
Just.
Justin87 is offline   Reply With Quote
Old 01-18-2017, 12:58 AM   #9
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 977
Default

Your protocol should produce a single peak regardless of its size. Profile #4 looks like adapter ligated (Y adapters) size selected shotgun fragments which shows a double peak in Bioanalyzer run (one usually twice the size of other one). That is because adapter ligated fragments migrate on Chip slowly than the non-ligated fragments. In this case double peak disappears after PCR because most of fragments are linear amplified adapter ligated fragments.

I think that your sample might have some staggered or single stranded fragments that anomalously migrate on the Chip giving the appearance of two peaks. Running the sample on gel should give a single peak.

It is possible that the large peak observed after size selection was present from the start but was masked by larger fragments.
nucacidhunter is offline   Reply With Quote
Reply

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 07:47 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO