Ancient DNA: Methods and Protocols (31 page)

M. Knapp
et al.

Table 3

Master mix for blunt-end repair (Protocol 1)

Volume ( m L)

Final concentration

Reagent

per sample

in reaction

Water (add to 20 m L)

7.6

Buffer Tango (10×)

4

1×

dNTPs (2.5 mM each)

1.6

100 m M each

ATP (10 mM)

4

1 mM

T4 Polynucleotide kinase

2

0.5 U/ m L

(10 U/ m L)

T4 Polymerase (5 U/ m L)

0.8

0.1 U / m L

Table 4

Master mix for adapter ligation (Protocol 1)

Volume ( m L)

Final concentration

Reagent

per sample

in reaction

Water (add to 19 m L)

10

T4 Ligase buffer (10×)

4

1×

PEG-4000 (50%)

4

5%

T4 Ligase (5 U/ m L)

1

0.125 U/ m L

master mix before adding T4 ligase and mix gently thereafter.

White precipitate may be present in the ligation buffer after thawing. Heat the buffer vial briefl y to 37°C and vortex until the precipitate has dissolved.

2. Combine the eluate from step 3 with 0.5 m L of dsAdapter_1

(50 m M) and 0.5 m L of dsAdapter_2 (50 m M). Mix thoroughly and spin down. Add 19 m L of master mix to obtain a total reaction volume of 40 m L and mix. Visually verify that all reaction components are mixed well. Incubate for 1 h at 22°C in a thermal cycler (see Note 4).

3. Purify the reaction over a Qiagen MinElute silica spin column according to the manufacturer’s instructions, but perform two PE washing steps. Elute in 25 m L 1× TE (without Tween-20).

Incubate the elution buffer on the silica membrane for 5 min before spinning it through (see Note 5).

19 Generating Barcoded Libraries for Multiplex High-Throughput Sequencing 163

3.2.3. Prepare Beads

1. Resuspend stock solution of MyOne C1 streptavidin beads by gentle mixing. Use 15 m L bead suspension (per sample). Add 100 m L 2× BWT buffer, mix by pipetting and place on magnetic rack for 1 min. After discarding the supernatant, repeat the previous step and elute the beads in 25 m L 2× BWT buffer.

3.2.4. Library

1. Add 25 m L eluate from step 6 to 25 m L bead suspension (from Immobilization

step 7). Incubate 15 min at room temperature and occasionally shake the tube/plate.

2. Pellet the supernatant using the magnetic rack and discard the supernatant.

3. Wash 4 times with 100 m L 1× BW buffer to fully remove unincorporated dsAdapter_1 and dsAdapter_1 dimers. Change tube/plate after wash 1 and 3. Remove the supernatant from the last washing step shortly before adding the fi ll-in master mix (see Note 6).

3.2.5. Adapter Fill-In

1. Prepare a fi ll-in master mix for the required number of samples.

Specifi c details of the composition of the reaction are provided in T
able 5
.

2. Resuspend the bead pellet in 50 m L fi ll-in master mix. Incubate at 37°C for 20 min in a thermal cycler.

3. Pellet the beads using the magnetic rack and discard

supernatant.

4. Wash the beads twice with 100 m L 1× BWT buffer. Completely remove the supernatant!

3.2.6. Library Elution

1. Add 20 m L 0.1× TE (without Tween 20) and boil at 95°C for 3 min.

2. Place on magnetic plate for 1 min and transfer supernatant into a fresh, preferably siliconized tube (see Note 7). The eluate is the single stranded, partly (Illumina Solexa) or fully barcoded (Roche 454) library with truncated adapters.

Table 5

Adapter fi ll-in master mix (Protocol 1)

Volume ( m L)

Final concentration

Reagent

per sample

in reaction

Water (add to 50 m L)

38

Thermopol buffer (10×)

5

1×

dNTPs (2.5 mM each)

5

250 m M each

Bst Polymerase (8 U/ m L)

2

16 U

164

M. Knapp
et al.

3.2.7. Library

1. Perform quantitative PCR (qPCR) using 1 m L of truncated Quantifi cation

library and quant_primer_F and quant_primer_R. This is to get an estimate of your copy number before amplifi cation with the extension primers to avoid over-amplifi cation of the libraries.

Check qPCR amplicon on gel to identify potential adapter dimers. For a detailed protocol see
( 11
) . As ancient DNA molecules often contain uracil as a result of postmortem cytosin deamination, it is essential for accurate quantifi cation to use a polymerase that can read across uracil, such as Amplitaq Gold (Applied Biosystems). For a list of suitable polymerases see
( 12
) .

3.2.8. Extension and

1. Determine the number of PCR cycles to saturation from the Amplifi cation of Libraries

amplifi cation plots of the qPCR.

2. Prepare an amplifi cation master mix. Specifi c details of the composition of the reaction are provided in T
able 6
.

3. Amplify the libraries in a thermal cycler to just below saturation to avoid the formation of heteroduplexes and other PCR

artifacts that may interfere with downstream applications.

The thermal profi le is as follows:

95°C for 12:00 min

94°C for 0:30 min

Repeat for
N
cycles as determined by qPCR

58°C for 0:30 min

72°C for 1:00 min

72°C for >10:00 min

10°C for hold

Table 6

Master mix for extension and amplifi cation of libraries (Protocol 1)

Final concentration

Reagent

Volume ( m L) per sample

in reaction

Water (add to 50 m L)

18

10× buffer (10×)

5

1×

25 mM MgCL

5

2.5 mM

2

dNTPs (25 mM each)

0.5

0.25 mM each

ext_ primer_F (10 m M)

1

0.2 m M

ext_ primer_R (10 m M)

1

0.2 m M

Amplitaq Gold

0.5

2.5 U

Template

19

19 Generating Barcoded Libraries for Multiplex High-Throughput Sequencing 165

4. Load 4 m L of the amplifi ed PCR products on a gel to check for potential adapter dimers. Then purify the reaction using the Agencourt AMPure XP DNA purifi cation kit according to the manufacturer’s instr
uctions (also see

( 3
) ). Elute and store

DNA in 20 m L 0.1× TE.

3.3. Protocol 2

The second protocol is designed for barcoding preamplifi ed multiplex PCR products, but can also be used for barcoding regular PCR products. It should be noted that the protocol was initially designed for and tested on Roche’s 454 platform
( 13
) , but should theoretically be compatible with Illumina’s solexa platform by interchanging the respective adapters and primers. Since the protocol uses preamplifi ed (or even fully amplifi ed) PCR product as template material, smaller volumes can be used than in protocol 1.

Therefore, all reaction volumes have been cut down to 30 m L reactions to be more cost effi cient.

3.3.1. Blunt-End Repair

1. Prepare a master mix for the required number of reactions.

Specifi c details of the composition of the reaction are provided in T
able 7 . Mix car
efully by pipetting up and down or fl icking the tube with a fi nger. Do not vortex after adding enzymes.

Keep the master mix on ice if not immediately used to maintain full enzyme activity.

2. Add 15 m L master mix to 15 m L sample (dissolved in EB, TE, or water; see Note 3) to obtain a total reaction volume of 30 m L and mix. Incubate in a thermal cycler for 15 min at 25°C followed by 5 min at 12°C (see Note 3).

3. Immediately purify the reaction over a Qiagen MinElute silica spin column (for PCR products shorter than 100 bp) or the

Agencourt AMPure XP DNA purifi cation kit (for PCR products longer than 100 bp) according to the manufacturer’s

instructions. Elute in 15 m L 0.1× TE + 0.05% Tween-20.

Table 7

Master mix for blunt-end repair (Protocol 2)

Volume ( m L) Final concentration

Reagent

per sample

in reaction

Water (add to 15 m L)

5.7

Buffer Tango (10×)

3

1×

dNTPs (2.5 mM each)

1.2

100 m M each

ATP (10 mM)

3

1 mM

T4 Polynucleotide Kinase (10 U/ m L)

1.5

0.5 U/ m L

T4 Polymerase (5 U/ m L)

0.6

0.1 U/ m L

166

M. Knapp
et al.

3.3.2. Adapter Ligation

1. Prepare a ligation master mix for the required number of reactions. Specifi c details of the composition of the reaction are provided in T
able 8 . Since PEG is highly viscous, vor
tex the master mix before adding T4 ligase and mix gently thereafter.

White precipitate may be present in the ligation buffer after thawing. Heat the buffer vial briefl y to 37°C and vortex until the precipitate has dissolved.

2. Combine the eluate from step 3 with 2

m L dsAdapter_1

(50 m M) and 2 m L dsAdapter_2 (50 m M) (see Note 9). Mix thoroughly and spin down. Add 11 m L master mix to obtain a total reaction volume of 30 m L and mix. Visually verify that all reaction components are mixed well. Incubate for 1 h at 22°C

in a thermal cycler (see Note 4).

3. Purify the reaction using the Agencourt AMPure XP DNA purifi cation kit according to the manufacturer’s instructions (also see
( 3
) ) and elute in 15 m L 0.1× TE + 0.05% Tween-20.

3.3.3. Adapter Fill-In

1. Prepare a fi ll-in master mix for the required number of samples.

Specifi c details of the composition of the reaction are provided in T
able 9
.

Table 8

Master mix for adapter ligation (Protocol 2)

Final concentration

Reagent

Volume ( m L) per sample

in reaction

Water (add to 11 m L)

4.25

T4 Ligase buffer (10×)

3

1×

PEG-4000 (50%)

3

5 %

T4 Ligase (5 U/ m L)

0.75

0.125 U / m L

Table 9

Adapter fi ll-in master mix (Protocol 2)

Final concentration

Reagent

Volume ( m L) per sample in reaction

Water (add to 15 m L)

8

Thermopol buffer (10×)

3

1×

dNTPs (2.5 mM each)

3

250 m M each

Bst Polymerase (8 U/ m L)

1

8 U

19 Generating Barcoded Libraries for Multiplex High-Throughput Sequencing 167

2. Add 15 m L master mix to 15 m L eluate from step 6 to obtain a total reaction volume of 30 m L and mix. Incubate in a thermal cycler for 20 min at 37°C (use heated lid).

3. Immediately purify the reaction using the Agencourt AMPure XP DNA purifi cation kit and elute in 25 m L 0.1× TE or EB

without Tween-20.

3.3.4. Library

1. Perform quantitative PCR (qPCR) using suitable qPCR

Quantifi cation

reagents (e.g., the HS SYBR ® Green qPCR Kit, New England (Optional Step)

Biolabs) following the manufacturer’s instructions and adding 1 m L of truncated library and quant_primer_F1 and quant_

primer_R1 (see Note 10). This is to get an estimate of your copy number before amplifi cation with the extension primers to avoid over-amplifi cation of the libraries. Run out the qPCR

amplicon on a gel to identify potential adapter dimers. For a detailed protocol see
( 11 )
. If primer dimers are visible on the gel, purify the reaction again using the Agencourt AMPure XP

DNA purifi cation kit.

3.3.5. Extension

If step 1 of Subheading 3.3.4
(above) was performed, determine and Amplifi cation

the number of PCR cycles to saturation from the amplifi cation of Libraries

plots of the qPCR. In the absence of quantifi cation results, perform 15 PCR cycles.

1. Prepare a fi ll-in master mix for the required number of samples.

Specifi c details of the composition of the PCR are provided in T
able 10 .

Thermal profi le:

95°C for 12:00 min

94°C for 0:30 min

15 cycles or
N
cycles as determined from

60°C for 0:30 min

optional qPCR ( see
Subheading 3.3.4 )

72°C for 0:40 min

72°C for 10:00 min

10°C for hold

2. Purify the reactions using the Agencourt AMPure XP DNA purifi cation kit. Elute and store the double-stranded libraries in 20 m L 0.1× TE + 0.05 % Tween-20.

3.3.6. Final Quantifi cation

1. Perform qPCR using, e.g., the HS SYBR ® Green qPCR Kit of Libraries

(New England Biolabs) following the manufacturer’s instructions and adding 1 m L of the double-stranded fi nal library and Amp_primer_F and Amp_primer_R (see Note 11). Run out

the qPCR amplicons on a gel to identify potential adapter dimers. For a detailed protocol see
( 11 )
. If primer dimers are visible on the gel, purify the reaction again using the Agencourt 168

M. Knapp
et al.

Table 10

Master mix for extension and amplifi cation of libraries (Protocol 2)