Ancient DNA: Methods and Protocols (28 page)

146

A.W. Briggs and P. Heyn

Fig. 1. The library preparation procedure. (i) Damage in ancient DNA leaves 5’ and 3’

overhangs and uracil (U) bases; (ii) USER enzyme removes and repairs uracil sites; (iii)
T4

PNK polymerase and
T4
polymerase phosphorylate 5’ ends (
black
) and repair overhangs; (iv)
T4
ligase attaches nonphosphorylated A and B adaptors to the fragments at one end only; (v)
Bst
polymerase fi lls in from the nick to complete adaptor attachment; (vi) Adaptor-complementary primers and
Taq
polymerase amplify the entire library to immortalize the sample ahead of direct sequencing or capture and sequencing.

4. 10× Thermopol buffer (NEB, Ipswich, MA).

5.
Bst
DNA polymerase large fragment, 8 units/

m L (NEB,

Ipswich, MA).

2.3. Primary Library

1. 10× Thermopol buffer (NEB, Ipswich, MA).

Amplifi cation

2. AmpliTaq Gold DNA Polymerase, 5 units/

m L (Applied

Biosystems, Foster City, CA).

3. dNTP mix (25 mM each of dATP, dTTP, dCTP, dGTP).

18 Preparation of Next-Generation Sequencing Libraries from Damaged DNA 147

4. Adaptor-specifi c PCR primer pair (recommended annealing temperature 60°C).

5. Siliconized 1.7-mL tubes (Sigma-Aldrich, St. Louis, MO, cat T3406-250EA).

2.4. Secondary Library

1. Phusion High-Fidelity PCR master mix with HF buffer

Amplifi cation

(Finnzymes/NEB, Ipswich, MA).

2. Adaptor-specifi c PCR primer pair.

3. Agilent DNA 1000 chip.

4. Agilent 2100 Bioanalyzer.

2.5. Library

1. DyNAmo™ Flash SYBR ® Green qPCR Kit (Finnzymes/NEB,

Quantifi cation

Ipswich, MA).

2. Adaptor-specifi c PCR primer pair.

3. TE buffer (10-mM Tris–HCl pH 8.0, 1-mM EDTA).

4. Tween-20.

5. qPCR machine.

All steps

1. MinElute PCR purifi cation kit (Qiagen, Hilden, Germany).

2. Molecular grade water.

3. Thermal cycler.

3. Methods

All aDNA works must be done in a dedicated cleanroom. Once the molecules are ligated to suitable adaptors the library can be handled outside the cleanroom. Still, one must be careful to avoid cross-library contamination, especially when handling PCR-amplifi ed libraries.

3.1. aDNA Repair

Damaged DNA such as aDNA can contain overhanging 5’ or 3’

ends and deaminated cytosines in the form of uracil bases. Two alternative repair reactions, A and B, are presented below. Reaction A excises uracil bases from the DNA, cleaves the resulting abasic sites, and repairs remaining overhangs to leave ligatable blunt ends.

Reaction B repairs overhanging ends but does not excise uracils.

Choose one of these reactions depending on the results desired.

Reaction A: End repair with uracil removal

1A. Prepare a reaction mix containing 5-m L 10× NEBuffer 2, 23-m L aDNA extract, and 17-m L molecular grade water. Mix reaction and briefl y centrifuge to collect the reagents at the bottom of the tube (see Notes 2–5).

148

A.W. Briggs and P. Heyn

2A. Add 2-m L T4 polynucleotide kinase and 3-m L USER enzyme (see Note 6). Mix by gently fl icking the tube and briefl y centrifuge to collect the reagents at the bottom. Incubate for 3 h at 37°C in a thermal cycler.

3A. Add 1-m L T4 DNA polymerase to the reaction, mix by gently fl icking the tube, and centrifuge briefl y. Incubate at 25°C

for 30 min in a thermal cycler. Proceed to step 1 in

Subheading 3.1
.

Reaction B: End repair without uracil removal

1B.

Prepare a reaction mix containing 5-m L 10× NEBuffer 2,

23-m L aDNA extract (see Notes 2–5), and 19-m L molecular grade water. Mix reaction and briefl y centrifuge to collect the reagents at the bottom of the tube.

2B.

Add 1-m L T4 polynucleotide kinase and 2-m L T4 DNA polymerase. Mix by gently fl icking the tube and briefl y centrifuge to collect the reagents at the bottom. Incubate for 30 min at 25°C

in a thermal cycler. Pr
oceed to step 1 in Subheading 3.1 .

1.
(For both repair reactions A and B)
Purify the reaction with a MinElute spin column. Load column by adding 150-m L buffer PB to the reaction, mix carefully by pipetting up and down several times, then transfer to the spin column. After binding and washing according to the manufacturer’s instructions,

elute DNA from the column in 15-m L buffer EB. After adding buffer EB to the column incubate for 1–2 min before

centrifugation, to improve DNA recovery.

3.2. Adaptor

Having repaired the DNA fragments, this step attaches universal
Attachment

adaptor sequences to the fragments in two steps; fi rst, the 5’ ends of the fragments are ligated to 3’ ends of the adaptors; second, the 3’ ends of the fragments are extended along the ligated adaptor strands to complete double-stranded adaptor attachment (see (iv)
and (v) in Fig. 1 ). W
e recommend including an extraction negative (blank) and a blank library control (water only) in the library preparation for quality control.

1. Prepare a reaction mix containing 20-m L 2× Quick ligase buffer, 1-m L 2.5 m M adaptors, 15-m L purifi ed repaired DNA, and 3-m L molecular grade water. Mix all components well by

pipetting up and down several times. After mixing the components, add 1-m L Quick ligase. Mix by fl icking thoroughly, and spin down briefl y to bring the mixture to the bottom of the tube (see Note 7).

2. Incubate the ligation reaction for 5 min at 25°C in a thermal cycler. Purify with a Qiagen MinElute column. Load column

by adding 160-m L buffer PB to the ligation product, mixing thoroughly by pipetting, and transferring to the spin column.

18 Preparation of Next-Generation Sequencing Libraries from Damaged DNA 149

After binding and washing according to the manufacturer’s

instructions, elute the DNA from the column with 15-m L EB.

After adding buffer EB to the column, incubate for 1–2 min before centrifugation to improve DNA recovery.

3. Prepare a reaction mix containing 5-m L 10× Thermopol buffer, 0.5-m L dNTP mix (25-mM each dNTP) 15-m L ligated and purifi ed DNA, and 27.5-m L molecular grade water. Mix the reaction and briefl y centrifuge down. Add 2-m L
Bst
DNA polymerase, mix by gently fl icking the tube and briefl y centrifuge down. Incubate at 37°C for 30 min in a thermal cycler, followed by 20 min at 80°C to inactivate the
Bst
polymerase.

Remove a 2 m L aliquot of the product for library quantifi cation with qPCR (for qPCR instr
uctions, see Subheading 3.5 ) (see

Note 8).

3.3. Primary Library

In this step, the entire library is PCR-amplifi ed using the universal
Amplifi cation

priming sites provided by the library adaptors. The reaction takes place in the same tube as adaptor fi ll-in without purifi cation, avoiding any loss of material (see Note 9). Immediate amplifi cation effectively immortalizes the library by making many copies of every original template molecule.

1. Prepare a 50 m L volume of “PCR addition mix” containing 5-m L 10× Thermopol buffer, 0.5-m L dNTP mix (25 mM each dNTP), 5 m L each adaptor primer (10-m L M stock), and 1-m L

AmpliTaq Gold DNA Polymerase (see Note 10). Mix all 50 m L

into the heat-inactivated 50 m L fi ll-in reaction. Transfer to a PCR machine and perform the following cycling conditions:

95°C

12 min

95°C

30 s

|

60°C

30 s

|

×12

72°C

1 min

|

72°C

3 min

2. Purify the amplifi ed product with a Qiagen MinElute spin column. Elute in 50-m L EB (see Note 11). Quantify the product with qPCR alongside the retained aliquot of unamplifi ed

library, to check for amplifi cation success (see Note 12)
.

3.4. Secondary Library

For most applications, higher-fold amplifi cation of the library will
Amplifi cation

be necessary, requiring a second library PCR. For example, array hybridization capture requires up to 20 m g of amplifi ed library product
( 7
) . For secondary amplifi cations, we recommend using a proofreading polymerase such as the Phusion enzyme described here, to reduce the occurrence of PCR errors in fi nal sequences.

150

A.W. Briggs and P. Heyn

1. Prepare a PCR at a total volume 100 m L containing 50-m L

2× Phusion HF master mix (Finnzymes/NEB, Ipswich, MA),

5 m L each adaptor primer (10-m M stock), and 10 m L of primary-amplifi ed library.

2. Transfer to a PCR block or thermal cycler and perform the following cycling conditions:

98°C

1 min

98°C

30 s

|

60°C

30 s

|

×10–20 depending on

desired yield ( see Note 13 )

72°C

1 min

|

72°C

3 min

3. Purify the amplifi ed product with a Qiagen MinElute spin column. Elute in 50-m L EB. Quantify the product with Nanodrop (less accurate) or an Agilent Bioanalyzer 2100 DNA 1000 chip (more accurate).

The repaired, immortalized, and quantifi ed DNA library is now ready for direct high-throughput sequencing or target enrichment with capture methods such as ar
ray hybridization ( 7
) , in-solution hybridization capture
( 8 )
, or primer extension capture
( 4
) followed by sequencing.

3.5. Quantifi cation

Conventional quantifi cation of unamplifi ed aDNA sequencing
of Library by qPCR

libraries is not generally possible by UV absorbance or fl uorescence measurements due to the very low amounts of material (below detection limits). While it is not strictly necessary to quantify the unamplifi ed library, as the secondary-amplifi ed (and sometimes primary-amplifi ed) library can be quantifi ed by UV or fl uorescence, it is usually very useful to know how much DNA the primary library contained, and whether the amplifi cation steps worked effi ciently.

To quantify libraries of low concentration, Meyer
et al.
( 6
) developed a qPCR quantifi cation method
( 21
) . Here we describe briefl y an updated version of that protocol. For further information on qPCR in aDNA work see Chap. 16 .

1. Prepare a dilution series of a sequencing library with known concentration, using 1× TE buffer to dilute. The range should be from 10 9 molecules/ m L to 10 2 molecules/ m L in tenfold dilution steps. This will be your standard for quantifi cation (see Note 14).

2. Prepare a qPCR Mastermix by adding molecular biology grade water and adaptor-PCR primers to the DyNAmo™ Flash

SYBR ® Green qPCR Kit according to the manufacturer’s

instructions.

18 Preparation of Next-Generation Sequencing Libraries from Damaged DNA 151

3. Dispense the mastermix into a qPCR-suitable plate.

4. Add 1 m L each template. Measure the standard dilution series at least twice. For quality control, include the unamplifi ed library, primary-amplifi ed library, secondary-amplifi ed library (if relevant), unamplifi ed extraction negative (blank) library, unamplifi ed blank library, and qPCR-blank, all in duplicates or triplicates. It might be necessary to include dilutions from the amplifi ed libraries (even up to 10,000-fold) to be in the quantifi cation range of the standard.

5. Perform qPCR with the cycling conditions following the instruction of the manufacturer, using the appropriate annealing temperature for the primer pair used.

6. Analyze the qPCR according to instructions from the qPCR

machine manufacturer (see Note 15).

4. Notes

1. The method we present here is compatible with any library adaptor sequences, although the oligonucleotide design may differ from manufacturers’ own kits due to differences in the ligation strategy. To make library A and B adaptors suitable for our ligation procedure, the adaptors must be designed as follows: for each double-stranded adaptor, the oligo that attaches to the 5’ end of the insert DNA should be ordered full-length, whereas the oligo that will attach to the 3’ end of the insert DNA should be ordered truncated to 12–14 nt, as fi rst

described in
( 22
) . Prepare the adaptors from lyophilized oligos as follows: dissolve each constituent oligo to 100 m M in TE

buffer; mix the two oligos of each adaptor 1:1, heat to 95°C

for 10 s then allow to cool to RT; mix the two resulting double-stranded adaptors 1:1, and dilute in TE 1:10 to form a fi nal 2.5-m M double-stranded adaptor stock.

2. Our library preparation procedure is compatible with aDNA extracted using any method, as long as DNA was not denatured during the extraction (adaptor ligation requires double-stranded molecules so denatured fragments cannot get into the library). Given the short lengths of many aDNA fragments,

this means keeping the temperature during extraction below ~60°C at all times.

3. The raw extract must be stored in a low retention tube, such as a Sigma-Aldrich siliconized 1.7-mL tube. DNA stored in regular polypropylene tubes can decrease dramatically in concentration over time due to DNA sticking to tube walls.

4. As much extract should be used as possible for aDNA library preparation, as the quality of the fi nal results depends on the 152

A.W. Briggs and P. Heyn

level of genomic coverage contained within the library.

However, we have not tested the protocol using more than

23 m L of pure extract in the 50 m L repair reaction. If more than 23 m L of pure extract is to be used, we recommend making multiple parallel libraries and pooling them prior to the primary amplifi cation reaction. However, if an extract will be diffi cult or impossible to replace, we recommend not using more than half of the extract in any single experiment, to keep some material behind in case something goes wrong.