This article has been updated to clarify that there are commercial kits available for NGS sample prep from single-stranded DNA.
NEW YORK (GenomeWeb) – Researchers from the Okinawa Institute of Science and Technology in Japan have developed a method for sequencing degraded DNA of limited quantity that cannot be amplified with conventional PCR.
The team demonstrated their method on museum samples, showing that with as little as 14 nanograms of highly fragmented DNA, they could do both low-coverage whole-genome sequencing and also RAD-tag sequencing in order to resolve the individual species. They published the protocol this month in PLoS One.
Senior author of the paper and assistant professor of ecology and evolution at OIST Alexander Mikheyev told In Sequence that the goal was to design a method that worked well on degraded, single-stranded DNA. While there are commercially available kits for degraded DNA, many of those kits require double-stranded DNA. One exception, however, is Swift Biosciences' Accel-NGS kit, which works with single-stranded DNA from degraded samples. In samples that only have single-stranded DNA, the kits often do an end repair step, which "uses endonucleases to chop up single-stranded bases, and a lot of information is lost," he said. "Studies have shown that there is a problem with these approaches having low library diversity."
Instead, Mikheyev's team designed an approach that would work directly with single-stranded DNA.
During sample prep, the researchers use a terminal transferase to attach a tail to the ssDNA and then add a riboguanadine. Terminal transferase tails are notoriously difficult to work with because they simply keep growing, Mikheyev explained, but the addition of a riboguanadine helps. The riboguanadine has a secondary structure that "terminates the tail after five bases, creating a 3' fragment that can be used to attach an adapter and create a second strand."
After that, the researchers prepare the molecule either for restriction site associated DNA tagging and sequencing or for shotgun sequencing.
For RAD-tag sequencing, after the riboguanadine is added and the DNA is primed with poly-C primer to create a second strand, the DNA molecule is digested with a restriction enzyme, after which sequencing adapters are added and the molecules are amplified.
For shotgun sequencing, after the second strand has been synthesized, the researchers prime the DNA with Illumina adapater sequence and the DNA is essentially ready for amplification and sequencing.
The OIST team is not the only group to work on a method for sequencing degraded, single-stranded DNA. Researchers from the Max Planck Institute for Evolutionary Anthropology last year published the protocol that they used when they sequenced the Denisovan genome to increase coverage from two-fold to 30-fold.
One difference between the two protocols is that the one developed by Max Planck uses an enzyme called CircLigase II, which adds biotinylated arms to the 3' ends of ssDNA.
Mikheyev said that while the protocol works well, the CircLigase enzyme is expensive, finicky, and labor intensive. Instead, he said his group wanted to design a protocol that usesmis off-the-shelf reagents and that would be amenable to automation since the team wants to apply the method to large numbers of environmental samples.
According to Marie-Theres Gansauge, lead author of the Max Planck protocol, the OIST method is a "clever idea" and the "whole scheme and the adapter design of the method seem to be well-conceived and convince me that this application probably will work on more difficult samples like ancient DNA."
However, she said that she still has questions regarding the addition of the 3' riboguanadines, noting that it is unclear how controllable that step is or how many riboguanadines are added during the reaction. "Undesirable mispriming of the poly-C primer could influence efficiency and data analysis after sequencing," she said. In addition, she said that the poly C stretches in the final library could affect base calling.
Mikheyev said that the researchers are continuing to tweak the protocol, with the most recent iteration available on the lab's website. One main difference, he said, is that the team recently added a phosphatase treatment step to the DNA fragments before using the terminal transferase. The phosphatase treatment boosts terminal transferase activity, which has resulted in around an order of magnitude increase in yield.
Now, he said, the team is using the method for a number of different projects, including a resequencing study of museum honeybee specimens to look at the bees' responses to parasitic mites and to look for evidence of selection and other genetic changes by studying samples pre- and post-exposure.
Mikheyev added that he does not intend to commercialize the method and that the protocol is freely available for other researchers to use.