Ancient DNA: Methods and Protocols (35 page)

6. Set up a master mix for the ligation of the biotinylated adapter, per reaction include:

(a)

10 m L H O.

2

(b)

4 m L T4 DNA ligase buffer (10×) (see Note 7).

(c)

4 m L PEG-4000 (50%).

(d)

1 m L adapter (200 m M).

(e)

1 m L T4 DNA ligase (5 U/ m L).

Vortex the master mix before adding T4 DNA ligase and mix

gently. Add 20 m L of master mix to each eluate from step 5 to obtain reaction volumes of 40 m L. Mix and incubate for 30 min at 22°C in a thermal cycler.

7. Purify the reaction using a MinElute column. Elute in 20 m L

EBT.

8. Set up a master mix for the
Bst
fi ll-in, include per reaction: (a)

14.1 m L H O.

2

(b)

4 m L ThermoPol reaction buffer (10×).

(c)

0.4 m L dNTPs (25 mM each).

(d)

1.5 m L
Bst
polymerase (8 U/ m L).

Add 20 m L of master mix to each eluate from step 7 to

obtain reaction volumes of 40 m L. Mix and incubate in a thermal cycler for 20 min at 37°C.

21 Target Enrichment via DNA Hybridization Capture

185

9. MinElute purify the reaction and elute in 20

m L of EBT.

Measure the concentration of DNA on a spectrophotometer.

The bait DNA can be stored at −20°C for several months.

Biotin can also be introduced during the polymerization of target regions up to 1 kb in size during regular PCR. For each reaction set up a master mix with:

1. 3.2 m L MgCl (25 mM).

2

2. 2 m L 10× PCR buffer.

3. 1 m L biotin-dUTP (100 m M) (see Note 8).

4. 0.2 m L dNTP mix.

5. 0.1 m L taq polymerase (5 U/ m L).

6. 3 m L primer mix (10 m M each).

7. 9.5 m L H O.

2

8. 1 m L template DNA.

After 5–12 min of denaturation at 95°C, run the PCR for 35

cycles of 30 sec 94°C, 30 sec at the respective annealing temperature, and 1 min at 72°C for elongation.

After a MinElute cleanup, measure the bait solution on a spectrophotometer and store it at −20°C.

3.1. Hybridization

1. Prepare between 100 ng and 1 m g of library (to have a 10 times excess compared to the bait) for each hybridization reaction in 200 m L tubes or wells of a 96-well plate (see Note 9).

2. Set up a hybridization master mix with the following fi nal concentrations:

(a)

1× hybridization buffer.

(b)

1× blocking agent.

(c)

10–100 ng of bait (to achieve a fi nal ratio bait:library of 1:10).

(d)

Blocking oligos (each 2 m M).

(e)

Water to 50 m L per hybridization reaction (accounting for library above).

3. Mix the master mix and add it to the library, resulting in 50 m L

hybridization reactions (see Note 10).

4. After denaturation of the mixture at 95°C for 5 min, carry out the hybridization rotating at 65°C in a conventional

hybridization oven (e.g., from SciGene) or in a thermal cycler (see Note 9). In the latter case, heat to 95°C for 5 min and then cool down to 65°C at 0.1°C/s.

5. Incubate at 65°C for 24 h or up to 48 h (see Note 11).

186

S. Horn

3.2. Immobilization

1. After hybridization, incubate the mixture with 5 m L magnetic
of Target-Enriched

streptavidin-coated beads for 20 min at room temperature

Library

(see Note 12).

2. Place the mixture into a magnetic rack to separate the magnetic beads from the supernatant (see Note 13).

3. Discard the supernatant, which contains the nontarget molecules.

4. Wash the beads 5 times using 1×BWT buffer, once in pre-warmed HW buffer at 50°C for 2 min, and once with 1×BWT.

5. Transfer the beads into a new tube and wash with 100 m L of TET.

6. Separate hybridized target molecules from the bait in 30 m L TE

by incubation at 95°C for 5 min in a thermal cycler. The eluate containing the sequencing library enriched for target DNA is ready for amplifi cation, quantifi cation, and sequencing.

3.3. Serial

1. After the fi rst hybridization capture, amplify the resulting
Hybridization Captures

library using the Phusion PCR master mix.

2. Purify the resulting amplicon with a MinElute column and use it in another round of hybridization capture starting at

Subheading 3.1
, step 1.

3.4. Amplifi cation,

1. Amplify the resulting library using the Phusion PCR master
Quantifi cation,

mix (see Note 14).

and Pooling Before

2. Quantify the enriched sequencing library with a spectropho-

Sequencing

tometer or by quantitative PCR.

3. Pool libraries of different samples (and negative controls, if applicable) in equimolar amounts for sequencing.

4. Notes

1. For hybridization on NimbleGen and Agilent arrays or when using the SureSelect in-solution kit, follow the protocols provided by the respective manufacturers
( 16, 17 )
.

2. The use of blocking oligonucleotides is not mandatory but may increase the percentage of sequencing reads that map to the desired target region.

3. Since blocking oligonucleotides will have a length of more than 30 bp, most companies only provide them HPLC purifi ed.

This prevents shorter oligonucleotides (aborted synthesis

products not reaching the full length) from being delivered along with the order. Special handling is not necessary.

21 Target Enrichment via DNA Hybridization Capture

187

Because the blocking oligonucleotides are combined in the

hybridization mixture, potential cross-contamination is not a problem.

4. Repetitive regions should be excluded from your bait. Those could capture large amounts of repeats present in the library and swamp your sequencing results.

5. The two complementary oligonucleotides for the generation of a biotinylated adapter can be of arbitrary sequence; an example is given in
( 14
) .

6. SPRI beads (Agencourt Ampure XP kit) can be used for

cleanup instead of MinElute columns if many samples have to be processed in parallel. See
( 18
) for a detailed description of SPRI bead usage.

7. If white precipitate has formed in the 10× DNA ligase buffer after thawing, warm the buffer to 37°C and vortex until the precipitate has dissolved.

8. Increasing the amount of biotin-dUTP in relation to dTTP in the biotinylating PCR may yield a higher number of functional bait molecules. Up to 50% of dTTP can be substituted with its modifi ed counterpar
t ( 19
) . This, however, will increase the cost of the experiment.

9. Enrichment in solution can be carried out for many samples in parallel in 96-well plates. These can be placed in a thermal cycler and should be incubated without rotation to minimize the chance of contamination between wells when improperly

sealed.

10. When carrying out in-solution enrichment in tubes, be sure to seal the tubes properly and tape them with laboratory fi lm (e.g., Parafi lm) for the 65°C hybridization step under rotation.

11. In my experience, the throughput of DNA hybridization capture can be increased by using 96-well plates instead of single tubes and by shortening the hybridization time to 24 h,

although the consequences of shortening the hybridization

time have not been evaluated fully
( 17 )
.

12. Dynabeads should not dry out; therefore remove buffers only immediately prior to the next pipetting step.

13. The addition of Tween (0.05%) to TE buffer facilitates the handling of streptavidin-coated magnetic beads. The beads

will assemble closer to the magnet of the rack and will stick less to pipet tips and tube walls.

14. In case the enriched library will be sequenced on the 454 platform, a lower total amount of library is required for sequencing compared to Illumina and SOLiD. Thus, proceed with quantifi cation of the library. Depending on the quantifi cation results, the amplifi cation step might not be necessary.

188

S. Horn

Acknowledgments

I would like to thank the Volkswagen foundation and the Max Planck society for funding and M Stiller for helpful comments on this manuscript.

References

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Science 325(5938):318–321

2. Green RE et al (2010) A draft sequence of

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333–334

Case Study: Enrichment of Ancient Mitochondrial

DNA by Hybridization Capture *

Susanne Horn

Abstract

In ancient DNA studies focusing on estimating population histories, genetic markers are sequenced from a large number of samples belonging to the same species. Targeting loci of interest using traditional PCR

can be time-consuming, in particular when samples are not well preserved and multiple overlapping fragments are required. Here, I describe the process of generating DNA libraries from ancient DNA (aDNA) extracts for high-throughput sequencing. I use a serial in-solution DNA hybridization approach with subsequent bead capture to enrich libraries for the target locus, in this case the mitochondrial control region of ancient beavers (
Castor fi ber
). The resulting sequencing reads are run through quality control fi lters to obtain reliable consensus sequences. Using these sequences, I construct a phylogenetic tree, which agrees with previously published data regarding phylogeographic relationships among beavers.

Key words:
Ancient DNA , Hybridization , Enrichment , High-throughput sequencing , Array capture , In-solution capture ,
Castor fi ber

1. Introduction

Estimating the demographic history of ancient populations requires sequencing the same genetic locus from multiple ancient DNA extracts, which often vary considerably in the quality and quantity of preserved DNA. The workfl ow of this approach includes designing primers to amplify short, overlapping regions (around 100–200

base-pairs including priming sites), and replicating PCR amplifi cations *
Note
: In the case study presented in this chapter, I describe the enrichment of target DNA from ancient DNA extracts using a hybridization-based method described in Chapter 21 . I discuss specifi c challenges associated with using this method with ancient samples, including the generation of suffi cient template DNA and the analysis of high-throughput sequencing data. For more information on the analysis of high-throughput sequencing data, see Chapter 23 .

Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_22, © Springer Science+Business Media, LLC 2012

189

190

S. Horn

to authenticate the resulting sequences and quantify DNA damage.