DNA sequencing is busting Moore’s Law by getting far cheaper, far faster than expected. But it is also getting far more sensitive. Researchers can sequence DNA samples 25 times smaller than they could a year ago.

“It is amazing, how much smaller it is all getting,” says HudsonAlpha Institute for Biotechnology Genomic Services Laboratory scientist Cynthia Hendrickson. HudsonAlpha is a non-profit institute that houses basic research labs and an international genome services center, and acts as an incubator for biotech start-ups.

For whole genome sequencing, in recent months, Hendrickson’s group has routinely gone from sequencing as little as one microgram of input to 100 nanograms. For work of less complexity, such as chromatin immunoprecipitation (investigating protein/DNA interaction), company protocols consistently direct the use of ten nanogram samples, but many labs have used much less. “In our lab, we have successfully gone as low as one picogram in tests,” Hendrickson says.

As recently as 2009, five-to-ten micrograms of material were still being recommended for some DNA or RNA sequencing. “Protocols were being released in 2007 for both Illumina and 454 asking for one-to-five micrograms of DNA. In 2009, 454 increased the input to five-to-ten micrograms for gel-size selected libraries,” explains Hendrickson.

But within the last year or so, many companies have developed far more sensitive reagents and protocols, allowing for the sequencing of samples in the hundreds of nanograms. Kits like New England Biolab’s NEBNext Ultra recommend inputs as low as five nanograms—although, cautions Hendrickson, when performing whole genome sequencing, customers must rely on their own sense of “how low you can go and still retain the complexity of the library (avoiding genome region losses). The lower the complexity of the sample, the less material you can use and retain all the material of interest. We have seen good results for 100 nanograms of human DNA. When we have enriched for only a small part of the human genome, as in ChIP-Seq, we can use much less. We can also use less for whole genome sequencing of species with smaller genomes, e.g. we can use less DNA to sequence E. coli than human, without losing complexity.”

Indeed, using the NimbleGen SeqCapEZ Human Exome v3 for exome (protein-coding DNA) capture, Hendrickson recently reported in a webinar that her group achieved similar success starting with 100 nanograms of genomic DNA—using the NEBNext Ultra—as they did when starting with 2.5 micrograms of genomic DNA using their standard protocol.

RNA can be sequenced accurately at even lower levels, as it is less complex, representing transcripts from only one to two percent of the genome (in the case of human). Hendrickson’s group prefers to use total RNA inputs of 100 nanograms. (Five percent or less of the total RNA is the mRNA they actually convert into library in human, so they are using about five nanograms of mRNA.) “But we can obtain good results with ten nanograms or less, or about 500 picograms of mRNA.”

The ability to sequence ever smaller swaths of genomic material matters, especially for clinical samples. Cell lines for basic research can be grown up in the lab in large quantities. But patient disease samples, coming from clinicians seeking biomarkers for diagnosis or drug compatibility, are often small. And clinical samples periodically must be divvied up by pathologists doing tests for different parties, which can include next generation sequencers, among others.

Furthermore, surgeons tend to use formalin-fixed paraffin embedding to preserve tissue biopsies, which can significantly degrade DNA. “Often, you really do need to use as little material as possible,” Hendrickson notes.

Streamlining clean-up is one key way library prep kit makers have lowered input requirements. In the case of Hendrickson and her initial trial of NEBNext Ultra, cleanup was halved. DNA sequencing involves multiple steps including end repair, A-tailing, adapter ligation, and polymerase chain reaction (PCR). “Originally there was clean up after every step, to remove buffers and enzymes. So after the end repair there would be clean up; A-tailing, clean up, and so on. In each of these cases there would be a physical transfer of the sample,” she says. Some of the sample would be lost.

But new kits minimize clean-up. New England Biolabs combines end-repair and A-tailing in one step. It also uses more efficient enzymes. The result: the reaction is not transferred from vessel to vessel, but stays put. There is effectively one washout, toward the end. Many DNA sample prep companies have started to offer variations on this approach. “You no longer worry you are washing away a bit of your sample every time you do clean up.”

Roche, for 454, developed one of the first library prep protocols involving successful removal of purification steps. Companies in the Illumina reagent market followed. New England Biolab’s Ultra was made for Illumina library prep methods, although there is some compatibility with some other platforms, like the Applied Biosystems’ 5500 Series SOLiD and Roche 454.

The bottom line, says Hendrickson, is that her group achieved “excellent” results using the NEBnext Ultra prep kit to sequence exomes. They used DNA inputs “far lower than we dared before.” They even attained greater yields of DNA after library prep, and they generated similar sequencing results. Finally, “the library prep was simpler and faster than our current method,” requiring fewer beads and sample transfers, and cutting labor by about an hour.

Moore’s Law dictates that computer technology improvements double every two years. DNA sequencing technology is clearly outpacing this, leading geneticists have noted. The first whole human genome was sequenced in 2003 after 13 years of work and an outlay of $3 billion. In January 2012, less than a decade later, Life Technologies produced the Ion Proton Sequencer. Life said its sequencer would, in a year, be decoding the whole human genome in a day for $1,000. Various labs are trying this out now.

The consumer genetics company 23andme periodically offers $999 whole exome sequencing.

The DNA sequencing market was worth over $1 billion in 2011, according to Decibo, a market research company.