TITLE: Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues
AUTHORS: Erin E Hahn, marina.alexander, Alicia Grealy, clare.holleley

[DESCRIPTION]
The widespread practice of formalin preservation has historically limited genomic analysis of archival museum specimens. 

Here we describe sample selection and extraction methods to recover genomic DNA sufficient to reconstruct complete mitochondrial genomes and achieve up to 20X nuclear genome coverage from formalin-preserved tissues. 

We describe in detail our approach to vetting specimen preservation quality as well as our procedure for processing tissues with hot alkaline lysis (adapted from Campos et al. 2012) coupled with phenol-chloroform extraction and a small-fragment optimised SPRI bead clean up protocol.

The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.


[BEFORE_START]
Managing Expectations

Our specimen vetting methods will identify unsuitable specimens and help to avoid unnecessary destructive sampling of unsuitable specimens. However, due to variation in preservation practices, we cannot completely guarantee that specimens identified as suitable will yield sequenceable DNA. 

We have successfully used these methods on liver, muscle, ovary and testis tissues as well as whole fish larvae and for samples as old as 1905. 

The DNA you extract from these specimens will be highly fragmented. In our experience, the mean fragment length is between 50 & 150bp. 

Sequencing Considerations

Amplicon-based sequencing approaches must be designed to target fragments in this range. 

De novo genome assembly from these sorts of samples is not recommended and care must be taken to select an appropriate reference genome for mapping. We have had success mapping of species to a reference of the same Genus, however, success will vary depending on evolutionary distance and reference quality.

For library preparation, we have found that kits designed to take fragmented input work very well. For example, the IDT xGen Cell Free and FFPE kit.

We highly recommend using a trimming-free read alignment approach such as kalign as implemented in the ngskit4b toolkit. See our manuscript in Molecular Ecology Resources for further details.

Capture-based sequencing methods have been successfully applied to formalin-preserved specimens and we encourage use of our extraction protocol as input for capture approaches. 

[STEPS]
SECTION: Specimen vetting
3. Measure the pH of the media. 


 

SECTION: Specimen vetting
1. Visually assess the specimen. If the specimen appears decomposed, consider selecting an alternative specimen. Generally, specimens with discernible internal organs tend to have higher likelihood of success. If the internal organs have been removed, this may indicate that the specimen had begun to decompose prior to fixation and will be unsuitable for DNA sequencing. Consult with your curatorial team as, in some cases, removal of the internal organs may have been conducted for alternative purposes that may not have influenced DNA preservation.


 

SECTION: Specimen vetting
2. From the jar containing your visually suitable specimen, take an aliquot of the specimen media into a 15 mL tube for down-stream measurement of residual formaldehyde and pH. If using a pH meter, you'll want to remove enough media such that you can adequately immerse the probe - typically at least 7 mL.

SECTION: Specimen vetting
4. Measure the residual formaldehyde concentration [F] of the media. 


 

5. Rate your visually well-preserved specimen for suitability against your sequencing objectives.

If the pH measures lower than 6 or the [F] measures greater than 10,000 mg/L, consider using an alternative specimen or limit your interrogation to small amplicon-based approaches.
If the pH is neutral and the [F] sits above 0 and below 10,000 mg/L, treat the specimen as formalin-fixed and well-preserved, thus suitable for broader-scale DNA sequencing, depending on extraction yield.
If the pH is neutral and there is no detectable formaldehyde, treat the specimen as ethanol-preserved. 
SECTION: Day 1 - Tissue Preparation
8. Pulverisation



 


6. Dissect at least 50 mg of your target tissue and remove to a 2 mL tubes containing 70% ethanol. 


 

SECTION: Day 1 - Tissue Preparation
8.1. Weigh and record the total tissue mass, subsampling to 50 mg if more tissue is available.

Note: More than 50 mg can be processed. Here we have limited it to 50 mg as we find this amount pulverises well without rupturing the tissueTUBE bag.
SECTION: Day 1 - Tissue Preparation
8.2. Transfer the tissue to an extra thick TT05 Covaris tissueTUBE bag and attach a 1 mL Covaris milliTUBE.


 


 

SECTION: Day 1 - Tissue Preparation
8.3. Dispense ~ 1L liquid nitrogen to a cryo dewar and flash freeze the tissueTUBE bag through immersion for approximately 30 sec.
 

SECTION: Day 1 - Tissue Preparation
8.4. Apply 2-3 level 6 impacts to the tissueTUBE with the Covaris CryoPrep, flash freezing the sample between impacts.


 

 

SECTION: Day 1 - Tissue Preparation
9. Tissue re-hydration and formaldehyde mop-up
SECTION: Day 1 - Tissue Preparation
9.1. Resuspend the tissue in cold 50% ETOH and transfer to a 15 mL tube using a wide-bore pipet tip. Rinse the bag with cold 50% ETOH to collect all material. Bring volume to 10 mL with cold 50% ETOH. Rock for 10 min at 4ºC.
SECTION: Day 1 - Tissue Preparation
9.2. Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold 30% ETOH. Rock for 10 min at 4ºC.
 

SECTION: Day 1 - Tissue Preparation
9.3. Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold milliQ water. Rock for 10 min at 4ºC.
SECTION: Day 1 - Tissue Preparation
9.4. Spin 10min at 750 x g 4ºC. Remove supernatant and resuspend tissue in 10 mL cold GTE buffer. Rock over night at 4ºC.
SECTION: Day 2 - Hot Alkaline Lysis
10. Spin the tube for 10 min at 750 x g 4ºC. Remove and discard all but 1 mL of the GTE buffer.
SECTION: Day 2 - Hot Alkaline Lysis
11. Resuspend the tissue in the 1 mL of retain GTE and transfer the tissue to a 2 mL screw-cap tube with a wide-bore pipet.
SECTION: Day 2 - Hot Alkaline Lysis
12. Spin the screw-cap tube for 10 min at 750 x g 4ºC. Remove the supernatant and add 500 μL Alkaline Lysis Buffer (0.1NaOH, pH 13; 1% SDS) to the pellet.

 


SECTION: Day 2 - Hot Alkaline Lysis
13. Secure the tube upright in an autoclave save container and autoclave at 120 ºC for 25 minutes.
 

 

SECTION: Day 2 - DNA Purification
14. Phenol:chloroform:isoamyl alcohol extraction
 

SECTION: Day 2 - DNA Purification
14.1. Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1) to the screw-cap tube of lysate, briefly vortex to resuspend and then rock for 5 min at room temperature.

SECTION: Day 2 - DNA Purification
14.2. Centrifuge the tube at maximum speed in a microcentrifuge for 5 min at room temperature.
SECTION: Day 2 - DNA Purification
14.3. Remove the aqueous phase to a new 2 mL tube and add 100 μL 10 mM Tris-HCl pH 8.0 to the organic phase. Rock the tube containing the organic phase for an additional 5 min.

 


SECTION: Day 2 - DNA Purification
14.4. Centrifuge the organic phase tube at maximum speed in a microcentrifuge for 5 min at room temperature. Combine the aqueous phase from this extraction with that recovered from the first extraction. Discard organic phases in the appropriate waste stream.
SECTION: Day 2 - DNA Purification
14.5. OPTIONAL: Add 1 μL RNase A to the extract and incubate at room temperature for 30 min.
SECTION: Day 2 - DNA Purification
14.6. Repeat the phenol:chloroform:isoamyl alcohol extraction.


 

SECTION: Day 2 - DNA Purification
15. Chloroform clean-up
SECTION: Day 2 - DNA Purification
15.1. Add equal volume chloroform and rock for 5 min at room temperature.
SECTION: Day 2 - DNA Purification
15.2. Centrifuge the tube at maximum speed in a microcentrifuge for 5 min at room temperature. Remove the aqueous phase to a new 2 mL tube.
SECTION: Day 2 - DNA Purification
15.3. Repeat chloroform extraction until the phenol is completely removed. Usually, we only perform one chloroform extraction and trust that the phenol is removed if we were able to remove the aqueous phase cleanly.
SECTION: Day 2 - DNA Purification
16. DNA concentration with SPRI beads
SECTION: Day 2 - DNA Purification
16.1. To each tube, add 1.5X volume small fragment-optimised bead solution and incubate at room temperature for 15 minutes.
SECTION: Day 2 - DNA Purification
16.2. Briefly spin the tubes and place them on a magnet for 10 minutes or until the solution is clear.
 

SECTION: Day 2 - DNA Purification
16.3. Remove the supernatant and wash the bead pellet twice with fresh 70% ethanol.
SECTION: Day 2 - DNA Purification
16.4. Briefly spin the tubes and return to the magnet. Remove remaining ethanol with a small volume pipette and allow the beads to dry for 30 seconds.
SECTION: Day 2 - DNA Purification
16.5. Add 30 μL 10 mM Tris-HCl, pH 8.0 and resuspend the beads by pipetting up and down.
SECTION: Day 2 - DNA Purification
16.6. Incubate the tube at 37ºC for 15 minutes then pellet the beads on the magnet once again for 15 minutes.
SECTION: Day 2 - DNA Purification
16.7. Transfer the supernatant containing your extracted DNA to a new tube.
SECTION: Day 2 - DNA QC
17. Measure the DNA concetration via Qubit and/or Tapestation.
 

 

7. Transfer and store the dissected tissue at room temperature until further processing.
SECTION: Spotlight video
18.