Issues with NGS Library Prep
Greetings Folks, I apologize if this is the wrong spot for this but I'd like to cast a net to see if I can get any additional thoughts or help.
I recently started a new job as NGS Library Prep Tech - sadly I had only begun training on this briefly at my last position but only got an introduction to Speed/Mag Bead clean up. I was hired because the lab is growing quickly and has had issues with organizational stuff in the past and that is my strong suit (my last position I did a lot of clonal DNA / miniprep stuff as far as the wet work went).
The person I was replacing at my new job was only there for two days and didn't really help a whole lot other than hand me a haphazardly written protocol and said "practice by cleaning ladders at different bead concentrations and running them on a gel."
Did that and was told they look good.
Fast forward to using actual samples: There were a set that needed to be redone because the final pool was lost. When I did my first qubit quants after the post PCR speed bead clean up I noticed that the quant concentrations were ~80% less than what they had been previously.
Today I have some remaining sample that I can push through PCR, my plan is to quant 8 out of the 53 samples of the pre / post PCR plate and then again after I do clean up.
As far as clean up goes I had been trying to do the whole plate at once, but I'm going to go back to just completing a column at a time to ensure my timing is better.
Are there particular spots in the Speed / Mag Bead clean up process that I should be aware that I could be washing away / losing DNA?
Do people have any tips on how to be more 'sure footed' in this process?
Ways that I can better practice and say "yup, I've got this!"?
Thanks for the help, and if this should be posted somewhere else please let me know, but as this is 'science' related I thought it fit best here.
Hey, I have done a ton of NGS preps and have a lot of experience with various mag bead clean ups so maybe I can help. Just to make sure we start on the same foot, are you using a specific kit or is this a more generalized cleanup protocol? Also what are your bead incubation times and how many washes are you doing?
Not a specific kit we are a super small lab that is doing genomics for conservation work.
To give you an idea - we're not buying TE buffer but making it in house and being as cost efficient as possible.
The PCR protocol is 3RAD.
The speed bead creation is based off of protocols in a paper:
Rohland N, Reich D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Research 22: 939-946.
Wash times & number of washes:
Post PCR Speed bead clean up is as follows:
a. Add 25 µL PCR H2O and 60 µL of beads
b. Vortex then let sit for 5 minutes
c. Put on magnet box for 1 minute
d. Remove liquid with pipette (set pipette at 120 µL)
e. Add 120 µL 80% ethanol ~60 seconds
f. Remove ethanol with pipette (set at 135 µL)
g. Remove remaining liquid with toothpick and remove from magnet, let beads dry until matte appearance.
h. Add 60 µL PCR H2O (After addition of PCR H2O, DNA is no longer bound to beads)
i. Vortex and let sit for 5 minutes
a. Add beads in 1.2 times the volume of Pippin Prep pool
b. Vortex then let sit for 10 minutes
c. Put in magnet box for 1-2 minutes
d.Remove liquid with pipette
e. Add enough 80% ethanol to cover the beads + 20% for ~60 seconds
f. Remove ethanol with pipette
g. Remove remaining liquid with toothpick and remove from magnet, let beads dry
h. Add 32 µL PCR H2O
i. Vortex and let sit for 10 minutes
The end goal is to run the pool on a Pippin prep and collect the sample that should be quanting >1ng/uL but I'm seeing ~0.2 -0.3 ng/uL
When you go to resuspend your beads after the ethanol wash are you noticing that they come into solution smoothly or are you seeing little flecks? Also just to be clear you are doing a clean up then pooling and cleaning the new pool immediately?
Some of the big weak points in the bead clean other than accidentally removing the beads during a wash (which you should be able to see) are that the beads are damaged. If the beads are fresh then damage can often happen by letting them over dry and crack. About how long are the beads drying for?
When you go to resuspend your beads after the ethanol wash are you noticing that they come into solution smoothly or are you seeing little flecks?
Also just to be clear you are doing a clean up then pooling and cleaning the new pool immediately?
About how long are the beads drying for?
If you are seeing little flecks then the beads are cracking. For pretty much all of the bead products I have used (Ampure, MagMax, PE) you can tell that the beads are damaged if there is any degree of flecks in the solution. Since you are resuspending in water and not TE you are probably safer on the less dry side. I would typically do 5 min dry cycles with only a single removal step (no toothpick or secondary tip) and with water you should be fine. I am wondering what the purpose of the second clean up is? What are you attempting to remove after pooling that was not removed in the initial post PCR clean?
Edit: just an anecdote, I have had multiple occurrences of letting a well with ~10-20uL of ethanol still in it be resuspended with 27.5uL water and still had normal post pcr concentrations (for us that could be in the 30-100ng/uL range, these were viral or bacterial whole genome samples).
Thanks for all the info @tyrny ; by little flecks do you mean do I see the beads clumping at all or do they come into a homogeneous solution easily - which I don't see any clumping when I resuspend with water.
I'm curious as to what the second clean up is and I'll follow up with that with others today.
The way I have always kind of described it is if the solution looks kinda like chocolate chip cookies. Light brown from resuspension of some but with darker specks from the others. Though I haven’t used the bead product you are describing and don’t know if they behave a little different than the other ones on the market. I hope you get good yields today! It can be so intensely frustrating when you get stuck troubleshooting a protocol, I wouldn’t wish it on anyone, lol.
Working through the process as we speak.
RE: Post pool clean up is to concentrate the DNA into 30uL because the pippin prep can only accept 30uL volume samples
Ah that makes sense. Good luck!
Things didn't go as I had hoped, I will return to the lab tomorrow to try again.
What I did observe during elution is that the speed beads didn't easily come off the walls when I pipetted in mH2O, but when it did it was quickly in solution.
I didn't observe any cracking during drying but these are in a 96w plate and a touch hard to see as clearly as I'd like.
I'm back at the start of digestion again tomorrow.
Wishing for a nanodrop for a bit more clarity on before / after clean up.
Any luck on getting the yields up?
The bad news that the post pcr clean up looked the same.
the good news is that the post pippin prep quant came back at 2.3ng/μL
Are you by any chance using Ampure Beads for the purification? I have some experience with them and the beads are unbelievably finicky and if you have any amount of ethanol left when you resuspend the beads in TE to elute you can end up with a contaminated sample which can then measure as being too low in concentration. I don't have the exact protocol on hand or memorized but there's like a 5-8min drying step we do on the beads after removing the ethanol to allow any remaining ethanol to evaporate prior to resuspending the beads in buffer.
Which form of sequencing are you using do you know? Like is it illumina or nanopore or something else?
Edit: actually one major thing we do differently is that we do the drying step without removing the beads from the magnetic rack and then do the resuspension off the rack. By transferring them you might be bringing along some of the ethanol.
We are using Sera-Mag SpeedBeads.
We are sequencing with Illumina.
Edit: I stand corrected. Looks like there are some people here now who can actually answer this. :)
Please label this comment offtopic so it doesn't detract from the on-topic discussion.
~science is the appropriate group on Tildes for this topic, but Tildes is still a pretty small community, so there may not be anyone here with the expertise needed to answer your questions. Which is why Reddit might be the better place to ask, since you could try posting in these presumably* related subreddits:
/r/askscience - /r/ngs - /r/bioinformatics - /r/genomics - /r/PharmacoGenomics
* - I didn't know what NGS was before this topic, so this is just a guess ;)
I figured I'd likely have to resort to reddit as well; I wanted to give it a shot here to see if anyone had any ideas.
I think NGS would be my go to and I'll see what if I can get help there as well.