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Aim

In a pilot experiment I tested three different genomic DNA extraction methods on the non-model organism Pieris mannii (southern small white; Insecta, Lepidoptera, Pieridae). The goal was to generate DNA fragments of ≥30 kbp average length for library preparation for PacBio SMRT long read sequencing (HiFi)).

Methods

The three methods/kits used were: Qiagen blood & tissue kit (column assay), Beckman & Coulter DNadvance (magnetic beads kit) and standard Phenol-Chloroform extraction.

Laboratory raised organisms in the prepupal stage (termination of larval growth phase, after last excretion of intestinal content, no chitinous exoskeleton) were harvested and frozen at -20°C. For the DNA extractions the head and the first two leg pairs (ca. 20mg) were cut and processed according to the kit/standard protocols with some modifications for high molecular weight DNA:

  • avoidance of freeze-thaw cycles
  • pestles were used for grinding the tissue (no liquid nitrogen)
  • inverting instead of vortexing
  • cell lysis and proteinase K digestion at 56°C and 300rpm overnight
  • planned: use of wide-bore pipette tips (impossible because of very long delivery waiting time)

Results summary

Method Avg. DNA fragment length (bp) range A260/280 range** A260/230 range** Nanodrop avg. conc. (ng/µl) Qubit avg. conc. (ng/µl)
Qiagen 1038 - 18366 2.07 - 2.12 1.77 - 2.15 1151.8 104.9
Magnetic beads 7350 - 12586 1.98 - 2.11 1.60 - 1.76 1048.7 155.8
Phenol-Chloroform 19552 - 27359 2.03 - 2.14 1.94 - 2.10 1389.2 215.4

*assessed with Agilent Femto Pulse
**Nanodrop results

The DNA fragments seem to be strongly sheared, the samples contaminated with organic compounds (especially the Magnetic bead ones) and the huge difference in concentration between Nanodrop and Qubit measurements may indicate RNA contamination.

Questions

How can DNA fragment length and purity be increased? Which are the most important measures? What life stage do you recommend using?

Ideas:

  • use of wide-bore pipette tips
  • shorten incubation time (cell lysis & proteinase K digestion)
  • use freshly eclosed imagines with soft exoskeleton to avoid slimy samples after cell lysis & proteinase K digestion (reason for contaminations?)
  • RNase A treatment
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    $\begingroup$ this is a fantastic question. very clear and comprehensive, informative, and using formatting effectively to drive home the point. I don't know enough to answer it though! $\endgroup$
    – Maximilian Press
    Jul 5, 2021 at 20:15
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    $\begingroup$ Welcome to Biology.SE! Strongly agree with MaximilianPress — I hope you will become a regular contributor. ——— I don't know how easily you can obtain samples, but you may need to try all of your suggestions (e.g. do a time course for the lysis/protK step) and see what works best. In addition, my understanding is that the Nanodrop is infamously prone to inaccuracy, so I would tend to trust the other results more. You might also want to think about doing something like contour-clamped homogeneous electric field (CHEF) electrophoresis to get a idea of the size distribution of your fragments. $\endgroup$
    – tyersome
    Jul 5, 2021 at 21:17
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    $\begingroup$ In absence of wide bore tips you can try cutting of some portion of the tip's front. Just ensure that the volumes you draw does not spill out. Obviously you won't be able to draw the maximum volume anymore. $\endgroup$
    – Roni Saiba
    Jul 6, 2021 at 2:35

2 Answers 2

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Insect samples are difficult to work with primarily because they contain a bunch of polysaccharides, like chitins, that are similar enough to nucleic acids that they can co-precipitate. Standard extraction procedures (e.g. Qiagen blood/tissue kits) do not handle this well without some modifications to get rid of these contaminants. The slimy aspect of your extractions is the result of these contaminants.

Back in my day (>20 years ago) handling plant specimens, which run into similar problems with poly-phenolics, extractions were performed using modified phenol-chloroform extractions. The specific form was a called a PCI (phenol-chloroform-isoamyl alcohol) extraction, with added B-mercaptoethanol and poly-vinylpyrrolidone in CTAB (cetyltrimethyammonium bromide) buffer.

I've had a quick search for Insect DNA extractions and found that similar protocols are in place for insects for long read-sequencing (see ref 4 in link). It looks like SDS with CTAB buffer is the way to go, followed by PC or PCI extraction.

In addition, measuring DNA and RNA concentrations by spectrometry (nano-drop etc) is unreliable - that method is great for telling you about DNA/RNA in protein solutions (the original purpose of the method), but is not really reliable enough for DNA/RNA quantitation for Gen 3 sequencing (or Gen 2 for that matter).

Instead, do as you have done, use a Qubit or other fluorescence method to quantitate and run your samples through a Bio-analyzer (Agilent) (high speed electrophoresis, with metrics on the sample added during analysis).

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  • $\begingroup$ Thank you very much for your input! $\endgroup$
    – CorinaPlus
    Jul 7, 2021 at 6:40
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I think @bob1's answer is good, and covers a lot of the bases.

One thing that I think is missing however is the use of a nuclear preparation as an initial step- my understanding is that this can help with a lot of the issues of phenolic/chitinous contaminants in difficult organisms like plants/fungi/insects. Here is one suggested protocol.

I would also recommend checking what's on protocols.io. Here is a protocol for insect HMW DNA prep, and here is a possibly relevant discussion.

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  • $\begingroup$ Those might be difficult on a whole organism - generally work well from cultured cells though, and I suspect that larvae will do quite nicely, as the OP is using. $\endgroup$
    – bob1
    Jul 23, 2021 at 0:56

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