Hi all! I will not be around while this poster session starts on Tuesday as I will be teaching a class, but I’ll pop back on here after 3 pm PST (11 pm UTC) to answer any of your questions!
Rowan French
Great poster, Brenna! I’m just about to start a cerambycid UCE project involving museum specimens, and I have a few questions about your methods:
1.) What were your typical input DNA concentrations for the samples from which you successfully sequenced UCE loci?
2.) Was there any association between specimen age and input DNA concentration or sequencing success?
3.) What was your success rate for UCE sequencing of museum specimens (i.e., did sequencing fail for some of your specimens?)
Thanks!
Rowan
Brenna Decker
Thanks, Rowan! Glad you’re getting involved with UCE work too! This was my first run-through of the UCE pipeline, so I may get better with selecting better specimens to start with and pipetting.
1) For the extraction Qubit values, we wanted over 10 ng/uL of original DNA, then we aimed to get 50 ng in a 100 uL stock solution to do the library prep – if we had a value of 20 ng/uL, we would use about 2.5 uL of DNA and 97.5 uL of water to get our stock solution. In some of the samples with lower concentrations of extracted DNA, I added a bit more if the library prep failed (so there were multiple attempts for some samples).
2) Age definitely plays a role in how much extracted DNA we can get, and how degraded that DNA is. However I also found that samples that were in malaise traps (most of mine) for extended periods had much lower extraction success rates. The older specimens I included, though they got about half of the UCE loci as others, were collected by net on a single day, so I think that played a bigger role in DNA quality.
3) I’ll start from the beginning on this one: I attempted extractions of about 130 samples in order to get 110 that gave some level of DNA. Of those 110 successful extractions, I was able to get 103 samples to PCR well enough through the library prep (though many more runs were done because I re-did many, both the PCR step and the whole library prep (sometimes twice) for a sample) – my sequencing lane had 110 total but the other 7 were non-Minagenia. So I had those 103 sent to sequencing, with 95 samples actually successfully providing sequences with my cutoff at 500 UCE loci or more – some samples only sequenced 20 loci, and those were from malaise traps.
My main take-a-way from this is, definitely budget for a few more specimens than your total target! I wish you luck on your future UCE endeavors!
~Brenna
Hi all! I will not be around while this poster session starts on Tuesday as I will be teaching a class, but I’ll pop back on here after 3 pm PST (11 pm UTC) to answer any of your questions!
Great poster, Brenna! I’m just about to start a cerambycid UCE project involving museum specimens, and I have a few questions about your methods:
1.) What were your typical input DNA concentrations for the samples from which you successfully sequenced UCE loci?
2.) Was there any association between specimen age and input DNA concentration or sequencing success?
3.) What was your success rate for UCE sequencing of museum specimens (i.e., did sequencing fail for some of your specimens?)
Thanks!
Rowan
Thanks, Rowan! Glad you’re getting involved with UCE work too! This was my first run-through of the UCE pipeline, so I may get better with selecting better specimens to start with and pipetting.
1) For the extraction Qubit values, we wanted over 10 ng/uL of original DNA, then we aimed to get 50 ng in a 100 uL stock solution to do the library prep – if we had a value of 20 ng/uL, we would use about 2.5 uL of DNA and 97.5 uL of water to get our stock solution. In some of the samples with lower concentrations of extracted DNA, I added a bit more if the library prep failed (so there were multiple attempts for some samples).
2) Age definitely plays a role in how much extracted DNA we can get, and how degraded that DNA is. However I also found that samples that were in malaise traps (most of mine) for extended periods had much lower extraction success rates. The older specimens I included, though they got about half of the UCE loci as others, were collected by net on a single day, so I think that played a bigger role in DNA quality.
3) I’ll start from the beginning on this one: I attempted extractions of about 130 samples in order to get 110 that gave some level of DNA. Of those 110 successful extractions, I was able to get 103 samples to PCR well enough through the library prep (though many more runs were done because I re-did many, both the PCR step and the whole library prep (sometimes twice) for a sample) – my sequencing lane had 110 total but the other 7 were non-Minagenia. So I had those 103 sent to sequencing, with 95 samples actually successfully providing sequences with my cutoff at 500 UCE loci or more – some samples only sequenced 20 loci, and those were from malaise traps.
My main take-a-way from this is, definitely budget for a few more specimens than your total target! I wish you luck on your future UCE endeavors!
~Brenna