User:Vanessa E Apkenas/Notebook/Historical songbirds

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Project Description

The purpose of this project is:

1. To develop optimized protocols for the sampling, extraction, and genotyping of historical songbird specimens, specifically Wilson's Warblers, with SNP assays on the Fluidigm EP1 System

2. To investigate temporal patterns in population structure over the past century and whether there are environmental/anthropogenic correlates

Notes on sampling

When sampling from historical bird specimens:

  • Wear gloves, use sterile exacto knife blades / scissors / etc. (bleach in between or use a new blade)
  • Use sterile collection tubes, etc.
  • If the bird is too small, sampling from the toe pads might cause too much damage. Instead, samples can be taken from the apterium on the bird's chest (more DNA will be attainable from larger tissue samples)
  • Store tissue samples in a low concentration DNA space, preferably an ancient DNA lab

Notes on DNA extraction Nov. 23-25, 2015

Extraction of DNA from historical songbird specimens was attempted with the following protocol (Nov. 23-25, 2015):

  • 16 individuals of Wilson's Warblers from the early 20th century (1904-1927, mostly 1910s), 2 tissue types each – body contour feathers (2), apterium skin (2x4mm)

Media:Wilsons_DickeyCollections_Nov2015.xlsx

1. Rinsed with 500ul 100% ethanol for 5 minutes, then 500ul 1X STE for 5 minutes

  • 1X STE recipe: 2ml 1M Tris, 1ml 5M NaCl, 0.2ml 0.5M EDTA; continue to 100ml with dH2O

2. Followed the Qiagen DNEasy Kit protocol with the following modifications:

Pre-incubation:

  • Added 40ul proteinase K per extraction (instead of 20ul)
  • Added 30ul 1M DTT per extraction (recipe: 50mg DTT + 450ul dH2O, increase recipe according to number of samples)
  • Incubated overnight

Post-incubation:

  • Half of the samples were spun with PCR purification spin columns and the other half were spun with the usual spin columns
  • AE buffer was pre-heated
  • Spin columns were incubated for 5-10 minutes before elution
  • First elution was 100ul, second was 100ul, both into separate tubes

This protocol was put together according to the following:

  • McCormack et al. 2015. Sequence capture of ultraconserved elements from bird museum specimens. Molecular Ecology
  • Fulton et al. 2012. Case study: recovery of ancient nuclear DNA from toe pads of the extinct passenger pigeon. In: Ancient DNA (eds Shapiro B., Hofreiter M.), pp. 29–35. Humana Press, New York, New York.
  • Mundy NI, Unitt P, Woodruff DS (1997) Skin from feet of museum specimens as a non-destructive source of DNA for avian genotyping. The Auk, 114, 126–129.

Recipes and advice given by Dr. Michael Sorenson

Extraction results (Rhodopsin amplification)

  • Agarose gel:

Media:gelmap.xlsx

Note: The expected size of the rhodopsin product is ~200 bp. Previous work with historical samples has shown a steep drop off in SNP genotyping success for SNP loci longer than 100-120 bp. Therefore the rhodopsin locus might be too long for successful amplification for historical samples.


  • E-gel (same order of samples with some unrelated samples at the end):

Note: E-gel requires dilution of PCR products, perhaps contributing to already faint bands

PicoGreen Results (ng/ul)

8A: -0.951242948

8B: -0.08401291

9B: -0.803419646

10A: -0.613713075

10B: -0.396905566

11A: -0.810810811

11B: -0.645741457

12B: 0.201778807

14A: 0.024390845

14B: -0.037202198

17: -0.305747863

18: -0.3895144

Note: These low values are not unusual for modern feather samples previously extracted in the lab.

Notes on DNA extraction Dec. 19-20, 2015

Extraction of DNA from 8 additional historical songbird specimens was attempted with the following protocol (Dec 19-20, 2015):

  • 8 individuals of Wilson's Warblers from the early-mid 20th century (1939-1954) from the Moore Lab of Zoology, all from apterium skin (most of the exposed apterium was taken, so approximately double the size compared to the previous round of specimens)

Media:Wilsons_MLZ_Extractions.xlsx

1. Rinsed with 500ul 100% ethanol for 5 minutes, then 500ul 1X STE for 5 minutes

  • 1X STE recipe: 2ml 1M Tris, 1ml 5M NaCl, 0.2ml 0.5M EDTA; continue to 100ml with dH2O
  • NEW: Tissues transferred to new tubes after rinses

2. Again, followed the Qiagen DNEasy Kit protocol with the following modifications:

Pre-incubation:

  • Added 40ul proteinase K per extraction (instead of 20ul)
  • Added 30ul 1M DTT per extraction (recipe: 50mg DTT + 450ul dH2O, increase recipe according to number of samples)
  • Incubated overnight

Post-incubation:

  • All 8 samples were spun with the usual spin columns
  • NEW: First flow-through of AL was replaced back into the spin column and re-spun to hopefully catch more DNA
  • AE buffer was pre-heated
  • Spin columns were incubated for 5-10 minutes before elution
  • NEW: First elution was 50ul, second was 50ul, both into separate tubes

Extraction results (Rhodopsin amplification)

  • Agarose gel:

Note: The negative control is only for the PCR reaction this time, not the extraction itself.

PicoGreen Results (ng/ul)

  • 1: 0.938267709
  • 2: 1.03067457
  • 3: 0.976582749
  • 4: 0.947283013
  • 5: 1.598638689
  • 6: 0.960805968
  • 7: 1.224503595
  • 8: 1.778944759

Notes on DNA extraction Jan. 5-6, 2015

Extraction of DNA from 8 additional historical songbird specimens was attempted with the phenol-chloroform protocol provided by the Moore Lab of Zoology (Jan. 5-6, 2015):

  • 8 individuals of Wilson's Warblers from the early-mid 20th century (1939-1954) from the Moore Lab of Zoology, all from apterium skin (most of the exposed apterium was taken)
  • The phenol chloroform protocol was altered in the following ways: 1) pre-digestion steps were identical to the Qiagen protocol (40 ul proteinase K and 30 ul DTT was added beforehand, digestion was overnight); 2) Day 2 steps 1-5 skipped; 3) Day 2 step 9 - used a small tip to scrape the top gel layer to the side of the tube so liquid can be poured into a new tube)
  • The extraction protocol was followed by a bead clean-up protocol pg 1-2 (to get rid of PCR inhibitors); this also excluded small DNA fragments (<100 bp)

Media:Wilsons_MLZ_Extractions.xlsx

Media:PhenolChloroform_Protocol.docx

Media:Beadcleanup_Protocol.docx

Media:Serapure_bead_recipe.pdf

Qubit Results (ng/ul)

  • 1: 7.10
  • 2: 10.9
  • 3: 6.28
  • 4: 6.62
  • 5: 3.74
  • 6: 6.31
  • 7: 10.1
  • 8: 4.19

Extraction results (not Rhodopsin)

  • Note: Samples 1-8 are in the bottom row; the top row are other samples run by W. Tsai

Extraction results (Rhodopsin amplification)

  • The control appears to be contaminated – note that this control is for the PCR only, not the extraction
  • Sample 3 evaporated during PCR, so this it likely not a failure

SNP Genotyping Jan. 13-14, 2016

  • Samples 8A - 12B from the Dickey Collections and all 16 samples from the Occidental Moore Lab of Zoology (extracted by Qiagen and phenol chloroform) were combined with other contemporary samples on a plate

Media:Platemaps_Jan13_2016.xlsx

PreAmplification Jan. 13, 2016

  • All historical samples were prepped in the aDNA lab and then amplified in the main lab according to the usual SNPtype protocol with the exception of additional cycles (30 instead of 13) during the PCR program
  • A preAmp control was included (only H20 and mastermix)

Chip run Jan. 16, 2016

  • A fresh SNP Assay stock plate was made (1/16/16) for this chip run
  • The following SNPtype genotyping protocol was used with a few adjustments (volume adjustments for additional mastermix; low TE instead of 2 uM Tris; etc.):

Media:SNPtype_protocol.pdf

Results summary

Average percent scorable SNPs per sample:

  • Dickey Collections -> 19.89%
  • Occidental MLZ Collections (Qiagen DNEasy extractions) -> 84.82%
  • Occidental MLZ Collections (Phenol chloroform extractions) -> 88.28%

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