Options When Starting Material is Limiting

Sometimes it is not possible to come up with the amount of DNA or RNA required for a standard Illumina library prep. We are frequently asked what the options are when there is just not enough sample available.

There are several kits on the market now that allow Illumina libraries to be prepared from minimal amounts of starting material. We have processed clinical samples, metagenomic samples, and samples from FFPE tissues that yielded extremely low amounts of RNA or DNA.

For RNA samples, we have generated linearly-amplified cDNA with the Nugen Ovation v2 kit. An advantage of this kit is that the amplification of rRNA is somewhat suppressed, increasing the percentage of usable data. Starting with sub-nanogram amounts of RNA, we are able to generate micrograms of cDNA. We’ve tested various library preparation methods with the amplified cDNA, and we have found that the Illumina TruSeq prep to work the best for us.

The Illumina Nextera system is an option available when DNA amounts are limiting. The Nextera XT DNA Sample Prep Kit requires exactly 1 ng of input material (best for plasmids or small genomes), and the Nextera kit DNA Sample Prep Kit requires exactly 50 ng of DNA. The library fragmentation is accomplished via transposon insertion events. We skip the normalization/denaturation portion of the protocol, and determine the quality and quantity of the libraries following our standard procedures. We have found that the library sizes tend to vary, and can be much wider than our traditional Illumina DNA libraries, but this is still a great option when there is very little material available.

Contact us if you have questions or would like additional information.