23 April 2014

ddRADseq

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ddRADseq round 2 summary

Samples

DNA was extracted from 3 ants each from 6 lab colonies of Aphaenogaster.

DNA concentration (ng/ul) and total mass in 40 ul

sample	colony	conc_extraction	total_dna
T1	EW205	42.4	1696
T2	EW110	60	2400
T3	2012A1	29.1	1164
T4	DG23	58	2320
T5	20130818-Mike	34.9	1396
T6	EW20 QR	35.2	1408

Combined total DNA ~ 1.0384 × 10⁴ ng

Double digestion

50 ul rxn volume following NEB protocol

Reagent	Volume (ul)
NlaIII	1
MluCl	1
DNA	40
CutSmart Buffer	5
H2O	4

3 hr digestion at 37°C.

Included “T7” Control sample of dH₂O only.

• Ln 1, Ap001, is genomic DNA. Note smear above largest marker and no/very little DNA within assay range.

• Ln 2 - 6 are double-digested samples. Looks like digest worked well for all samples as concentration in the 180 - 220 bp window ranged from 0.84 - 3.64 ng/ul.

AMPure Bead Purification

• added 75 ul AMPure Beads to the 50 ul rxn. Incubated 5 min room temp
• 2 min on magnetic plate. removed solution
• added 200 ul 70% ethanol (fresh). removed. repeat
• off plate, added 40 ul H₂O. 1 min
• on plate 2 min. transfered solution to new plate

Quantified DNA using Qubit.

DNA concentration (ng/ul) of samples after DNA extraction and after double digestion

sample	conc_extraction	conc_digestion
T1	42.4	10.9
T2	60	24.5
T3	29.1	5.19
T4	58	10.2
T5	34.9	5.82
T6	35.2	2.1
T7		0.13

• DNA recovery is low, only 20.7674% on average.
• Positive correlation between the input DNA and DNA after digestion.
• T7 is the blank control - note low-level DNA contamination.

## 
##  Pearson's product-moment correlation
## 
## data:  conc_extraction[1:6] and conc_digestion[1:6]
## t = 2.712, df = 4, p-value = 0.05344
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  -0.0195  0.9777
## sample estimates:
##    cor 
## 0.8048

## pdf 
##   2

Ligation

Input 100 ng total DNA from each sample for ligation. Volume of each sample to use as input for ligation reaction:

Sample	Volume DNA (ul)
T1	9.2
T2	4.0
T3	19.3
T4	9.8
T5	17.2
T6	47.6
T7	40.0

For each sample, combined DNA with adapter and T4 ligase. Determined concentration of adapters using ligation molarity calculator. Made fresh working stock of the universal adapter P2. Rather than make a working stock for each adapter P1, just used 0.5 ul of adapters P1.1 - P1.7 for samples T1 - T7, respectively. Made master mix of universal adapter P2 following worksheet.

Reagent	Volume (ul)
DNA	see above
T4 mix	4
P1 (4uM)	0.5
P2 mix	3
H2O	to 50 ul

DNA concentration (ng/ul) of samples after DNA extraction and after double digestion.

sample	Digestion conc	Ligation conc
T1	0.32	1.12
T2	2.9	0.28
T3	1.5	0.07
T4	0.9	1.78
T5	1.4	0.99
T6	0.7	5
T7		1.7

No correlation between Bioanalyzer concentrations for samples before and after ligation.

## 
##  Pearson's product-moment correlation
## 
## data:  bioan_digestion[1:6] and bioan_ligation[1:6]
## t = -1.173, df = 4, p-value = 0.3058
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  -0.9340  0.5185
## sample estimates:
##    cor 
## -0.506

## pdf 
##   2

Size selection

• Pooled 40 ul of each ligation in single 1.5 ml tube, leaving ~ 10 ul for diagnostics.
• AMPure bead purification - modified rack method using 96-well magnet plate
• Total pool volume 320 ul.
• 480 ul AMPure beads
• Two washes with 500 ul 70% EtOH
• Eluted in 30 ul H₂O
• Size selection
• ran gel for 2.5 hrs at 120V.
• To size select (1) 200+-20 bp, (2) 300+-20 bp, (3) 400 +- 20bp, had to account for 76 bp of adapter sequence. Cut gel pieces 250 - 300 bp, 350 - 400 bp, 450 - 500 bp

• QIAquick purification, eluting in 12 ul H₂O

DNA concentration (ng/ul) of pooled samples for different gel size extractions.

Sample	descrip	Conc
P1	Pooled - size 200	0.18
P2	Pooled - size 300	0
P3	Pooled -size 400	0.01

Very little to no DNA in each pooled size selection.

Possible places DNA lost

• pooling
• gel extraction