Welcome back! Last quarter we discussed why Next Generation, or Massively Parallel, Sequencing is the next big thing in the world of Molecular Diagnostics. The sensitivity, the depth of coverage and the ability to interrogate many different areas of the genome at the same time were just a few of the benefits of these types of assays. Next, I would like to describe a couple different methods of library preparation, which is the first step necessary to run an NGS assay.
First of all, let’s define “Library.” I find this is the most common question technologists new to this technology ask. Essentially, a library is a specimen’s collection of amplicons produced by the assay that have been barcoded, tagged with appropriate platform adapters and purified. These will serve as the input for the next part of the NGS workflow, clonal amplification (the topic of next quarter’s blog!). How these libraries are prepared differ depending on platform (i.e, Ion Torrent vs. MiSeq), starting material (RNA vs. DNA), and type of assay (targeted amplicon vs. exome).
Before we begin the library prep discussion, a note about the input specimen. The DNA must be quantitated using a method that is more specific than spectrophotometry – it must be specific for double-stranded DNA. It will lead to an overestimation of the amount of DNA in the specimen, which will lead to over-dilution and consequently, lower quantity of final library. Real-time PCR and a double-stranded kit with fluorometry are two examples of assays that will give accurate concentrations of double-stranded DNA.
Our lab has begun using NGS for some of our oncology assays, so I will focus on the two types we perform currently, but keep in mind, there are many other types of assays and platforms.
The assay we use for our Ion Torrent platform is a PCR amplicon based assay. The first step is to amplify up the 207 regions over 50 genes that contain hotspots areas for a number of different cancer types. This all occurs in one well for each specimen. Once those areas are amplified, the next step is to partially digest the primer sequences in order to prepare the ends of amplicons for the adapters necessary for the sequencing step. As shown in the figure above, two different combinations of adapters may be used. The top one, listed as the A adapter (red) and the P1 adapter (green), would be used if only one specimen was to be sequenced on the run. The A and P1 adapters provide universal priming sites so that every amplicon of every sample can be primed with the same primers, rather than having to use gene specific primers each time. The second possibility is listed below that, with the same P1 adapter (green) and a Barcode Adapter labeled X (red and blue) – it still contains the A adapter necessary for sequencing (red), but it also contains a short oligonucleotide sequence called a “barcode” (blue) that will be recognized during the analysis step based on the sequence. For example, Barcode 101’s sequence is CTAAGGTAAC – this will be assigned to specimen 1 in the run and all of the amplicons for that specimen will be tagged with this sequence. Specimen 2 will have the barcode 102 (TAAGGAGAAC) tag on all of its amplicons. During analysis, the barcodes will be identified and all of the reads with the 101 sequence will be binned together and all of the reads with the 102 sequence will be binned together. This allows many specimens to be run at the same time, thus increasing the efficiency of NGS even more. Lastly, the tagged amplicons are purified and normalized to the same concentration.
The assay we use for our MiSeq platform is a hybridization followed by PCR amplicon based assay. The first step is to hybridize probes to 568 regions over 54 genes that contain hotspots for a number of different cancer types. This occurs in one well for each specimen. Once the probes have hybridized, the unbound probes are washed away using a size selection filter plate. Next, the area between the probes is extended and ligated so that each of the 568 amplicons are created. These are then amplified in a PCR step using primers that are complimentary to a universal priming site on the probes, but also contain adapters plus the two indices required for paired end sequencing (the Ion Torrent platform utilizes single-end sequencing – this will be discussed in the sequencing portion in an upcoming blog!). As in the previous method, after PCR, these tagged amplicons are purified and normalized to the same concentration in preparation for the next step – clonal amplification.
Stay tuned for next quarter’s post – clonal amplification!
-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.