With the tidal wave of next-generation sequencing sweeping through the landscape of biomedical sciences and technology, researchers are inundated with a flood of data.
Yet the data will only be as useful as the quality of the libraries from which it is generated, and can be no more informative than the use to which it is put.
Researchers at the “International Plant & Animal Genome” (PAG) conference presented their insights and opinions on such issues as how to prepare a DNA library from limited, degraded, or even an overabundance of material, as well as how best to piece together the information once it’s obtained.
When there is a lot of high-quality DNA to start with, it’s relatively easy to make a good sequencing library. Yet in too many cases only a limited quantity is available. New England Biolabs (NEB) has optimized its new NEBNext®Ultra DNA Library Prep Kit to address this issue—“you can use it with as little as 5 ng of human genomic DNA to make a library, and the quality of the library is very good,” said Pingfang Liu, Ph.D., application and product development scientist.
Another advantage for this kit is that it can be used with both intact DNA and fragmented DNA, like FFPE-preserved biopsies or circulating plasma DNA, she said, adding that other NGS library preparation kits on the market claiming facility with low DNA input require that DNA to be intact. “We have a lot of customers with precious samples, and they have only one shot to make a library.”
Being a company that specializes in enzymes, NEB focused on optimizing the enzymes and conditions found in the library prep kit. For example, it developed a polymerase for the PCR amplification with very good efficiency with all types of amplicons—including GC-rich and AT-rich—which boasts about 100-fold greater fidelity than Taq. The cleanup step between different enzyme reactions was also minimized, “because with a small amount of DNA, if you need to transfer from one tube to another tube you tend to lose a lot of your substrate,” Dr. Liu said.
The ligase master mix has been specially optimized to ligate the types of ends that are present in Illumina library prep methods, and at this point the kit is being supplied with adapters for Illumina library prep. It is compatible with the workflows of other sequencing platforms such as the Applied Biosystems’ 5500 Series SOLiD and Roche 454 platforms that use single-base TA overhangs as well, “although the sequences of the actual adapters are different between the different platforms,” she said.
Dr. Liu discussed results from a collaboration with Cynthia Hendrickson, Ph.D., of the Genomic Service Lab at HudsonAlpha Institute for Biotechnology, on exome capture. Using the NimbleGen SeqCapEZ Human Exome v3, Dr. Hendrickson was able to obtain virtually the same results starting with only 100 ng of genomic DNA using the NEBNextUltra DNA Library Prep Kit as with 2.5 µg genomic DNA using a traditional protocol.
This is all the more impressive when keeping in mind that “you will lose 99% of your library because the human exome is only 1% of the whole genome,” Dr. Liu remarked.