06 December 2013

ddRADseq and restriction enzymes

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Wed - Fri notes



Ground about 40 Aphaenogaster in liquid nitrogen with mortar and pestle. Transferred to 1.5ml screw-cap tube with 500ul Buffer ATL. Added 1 scoop zirconium silicate beads. Bullet Blend for 3 minutes. Centrifuged 1 minute top speed. Added 50ul Proteinase K. Vortexed. Incubate overnight at 50C.

Proceed with Qiagen DNeasy extraction tomorrow.


Golan, D. & Rosset, S. (2011). Accurate estimation of heritability in genome wide studies using random effects models. Bioinformatics, 27, i317–i323.

However, the method suggested by Yang et al. (2010) might carry some hidden pitfalls. Replacing the correct genetic correlation matrix by a different matrix estimated from the data as if the latter matrix were the correct matrix is unfounded statistically. Indeed, using the A* matrix as the variance–covariance matrix introduces two additional sources of variance to the estimation. One is the error originating from the element-wise difference between A* and G (the noise from the non-causal variants) and one stems from the built-in variance of the estimated β. The former in particular is highly sensitive to the proportion of causal SNPs, as our simulations have demonstrated: with few causal SNPs, the noise in A* overwhelms the signal and estimation accuracy in the approach of Yang et al. (2010) deteriorates significantly.


climate cascade

ddRAD protocol - putting together list for about 500 samples

Oligo ordering

T4 ligase


Great concentration of DNA from DNeasy Aphaenogaster extraction!

elution 1 - 13.8 ng/ul * 200 ul = 2760 ng

elution 2 - 8.7 ng/ul * 200 ul = 1740 ng

total of about 4.5 ug DNA

Set up two more extractions with 30 ants each.



rm doesn’t accept regular expression as an argument. Use this command instead

ls | grep -P "yourstring$" | xargs -d"\n" rm


Debugging ApTran_assemble.R script…

climate cascade

Supplies for ddRADseq - order list on workstation in home/projects/climate-cascade/protocols/ddRAD_ordering_20131209.xlsx

Automatic size selection of DNA fragments requires Sage Sciences system.

Pippin Prep 90bp to 2kb $9,900

Blue Pippin 90bp to 50kb $15,000

Consumables cost the same - $.84/sample according to ddRAD protocol.

Either will have to do gel excision or find alternate method for DNA fragment size selection.


Two more DNA extractions of 30 Aphaenogaster each.

For each sample, did 1 elution of 200 ul and a second of 100 ul into the same tube.

ApDNA.2 - 10.1 ng/ul * 300 ul = 4.2 ug

ApDNA.3 - 6.2 ng/ul * 300 ul = 1.8 ug

combined two elutions from first extraction

ApDNA.1 - 10.1 ng/ul * 400 ul = 4 ug

TOTAL = 10 ug DNA

Right at minimum level for PacBio - do one more extraction, pool together and ship to Delawarefor sequencing! They will evaluate if fragment size is in fact greater than 20kb as promised by Qiagen for PacBio sequencing.

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