Blog-author: Sandeep Venkataram is a grad student in Dmitri Petrov’s lab.
By Sandeep Venkataram – Modern humans have been evolving independently of our nearest living relatives, chimpanzees, for over 7 million years. To study our evolutionary history since this divergence, our major source of information is from the fossilized remains of our ancestors and closely related species such as Neanderthals. Physiological information from the remains can tell us a lot about human evolution, but the majority of the information is locked up in the tiny amounts of highly degraded and fragmented DNA left from the specimen.
Studying ancient DNA (aDNA) from bones is extremely challenging. Each sample contains not only the fossil’s DNA, but contaminating DNA from enormous numbers of microbes and other organisms. There is also the possibility of human contamination from handling the fossil. Since the contaminants have much higher quality DNA than the endogenous sample, simply processing a raw DNA extract of the sample typically yields sequencing results with less than 1% aDNA. Sequencing such a sample is incredibly inefficient, as less than 1% of the sequence is actually useful and only the best funded studies can afford the sequencing costs to generate a high coverage genome. Therefore, most aDNA studies tend to focus on mitochondrial DNA using targeted capture methods, or PCR amplicon sequencing. This reduces wasted sequence capacity, but greatly limits the amount of information obtained from the sample.
Getting the most out of ancient DNA
Meredith Carpenter, who is a postdoc here at Stanford, and her colleagues have developed a novel method called whole genome in-solution capture (WISC) to purify aDNA from next-gen sequencing libraries generated from fossil DNA samples. The authors make use of RNA bait technology (Figure 1), which uses RNA complementary to the targeted DNA that has been synthesized using biotinylated nucleotides (Gnirke et al 2009). The RNA can be hybridized to the DNA pool, and purified from solution by linking the biotin to commercially available purification beads. By then eluting the hybrids from the beads and removing the RNA from solution, one can greatly enrich for the targeted DNA. Previous methods using RNA baits required synthesizing DNA strands on microarrays that are complementary to the RNA, then inducing transcription in vitro to generate the RNA bait library. This methodology has been successfully used to capture the entirety of chromosome 21 (Fu et al 2013) from human fossils, but is not cost effective for producing a bait library that covers the entire human genome.
Meredith Carpenter and coauthors circumvent this by mechanically fragmenting human genomic DNA and use blunt end ligation to attach adapter sequences containing an RNA polymerase promoter. This modified DNA library can then be used to generate the biotinylated RNA bait library via in vitro transcription, after which the RNA library is purified using standard protocols. The authors also generate non-biotinylated RNA complementary to the adapter sequences present on every DNA fragment in the library, to block nonspecific binding to the adapters. The bait and block RNA libraries are hybridized to the aDNA libraries, purified using beads to select only the aDNA fragments and sequenced. The cost of their enrichment method is estimated at $50 per sample, and is accessible to most labs conducting aDNA genomic studies.
WISC greatly enriches for ancient DNA across a variety of samples
The authors tested this method on a variety of aDNA libraries prepared from both high quality and low quality samples, including hair remains, teeth and bones, and fossils from tropical and more temperate regions, which can greatly influence DNA quality. They sequenced the libraries both before and after WISC, and found a 3-13x increase in the number of uniquely mapped reads after using WISC. In addition, most of the unique reads in the enriched library are sequenced with five million reads in both hair and bone samples. WISC allows most of the endogenous sequence to be read from dozens of aDNA samples in a single lane of Illumina HiSeq, opening the possibility of sequencing the millions of fossils in museums and collections around the world.
Dr. Carpenter says they are now focusing on adapting the method to removing human DNA contamination from microbiome sequencing projects with promising preliminary results, as well as applications in forensics and studying extinct species. As WISC generates the RNA bait library from genomic DNA of an extant relative instead of synthetic DNA arrays, bait libraries can be prepared regardless of whether the genome of the organism that is the source of the bait library is known. Combined with recent advances in aDNA library construction methods (Meyer et al 2012), WISC promises to make sequencing of contaminated and degraded samples widely accessible.
Carpenter et al (2013) Figure 1. Schematic of the Whole-Genome In-Solution Capture Process
To generate the RNA “bait” library, a human genomic library is created via adapters containing T7 RNA polymerase promoters (green boxes). This library is subjected to in vitro transcription via T7 RNA polymerase and biotin-16-UTP (stars), creating a biotinylated bait library. Meanwhile, the ancient DNA library (aDNA “pond”) is prepared via standard indexed Illumina adapters (purple boxes). These aDNA libraries often contain <1% endogenous DNA, with the remainder being environmental in origin. During hybridization, the bait and pond are combined in the presence of adaptor-blocking RNA oligos (blue zigzags), which are complimentary to the indexed Illumina adapters and thus prevent nonspecific hybridization between adapters in the aDNA library. After hybridization, the biotinylated bait and bound aDNA is pulled down with streptavidin-coated magnetic beads, and any unbound DNA is washed away. Finally, the DNA is eluted and amplified for sequencing.
Paper author Meredith Carpenter is a postdoc in Carlos Bustamante’s lab.
References
Fu, Q., Meyer, M., Gao, X., Stenzel, U., Burbano, H. a, Kelso, J., & Pääbo, S. (2013). DNA analysis of an early modern human from Tianyuan Cave, China. Proceedings of the National Academy of Sciences of the United States of America, 110(6), 2223–7. doi:10.1073/pnas.1221359110
Gnirke, A., Melnikov, A., Maguire, J., Rogov, P., LeProust, E. M., Brockman, W., Fennell, T., et al. (2009). Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nature biotechnology, 27(2), 182–9. doi:10.1038/nbt.1523
Meyer, M., Kircher, M., Gansauge, M.-T., Li, H., Racimo, F., Mallick, S., Schraiber, J. G., et al. (2012). A high-coverage genome sequence from an archaic Denisovan individual. Science (New York, N.Y.), 338(6104), 222–6. doi:10.1126/science.1224344
Update: The second paragraph originally talked about fossils, but it should have been bones (now corrected). A fossil is mineralized and would not yield aDNA.
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