To facilitate multiplexing, different barcoded adapters can be used with each sample. Alternatively, barcodes can be introduced at the PCR amplification step by using different barcoded PCR primers to amplify different samples. High quality reagents with barcoded adapters and PCR primers are readily available in kits from many vendors. However, all the components of DNA library construction are now well documented, from adapters to enzymes, and can readily be assembled into “home-brew” library preparation kits.
An alternative method is the Nextera DNA Sample Prep Kit (Illumina), which prepares genomic DNA libraries by using a transposase enzyme to simultaneously fragment and tag DNA in a single-tube reaction termed “tagmentation” (Figure 2)(22). The engineered enzyme has dual activity; it fragments the DNA and simultaneously adds specific adapters to both ends of the fragments. These adapter sequences are used to amplify the insert DNA by PCR. The PCR reaction also adds index (barcode) sequences. The preparation procedure improves on traditional protocols by combining DNA fragmentation, end-repair, and adaptor-ligation into a single step. This protocol is very sensitive to the amount of DNA input compared with mechanical fragmentation methods. In order to obtain transposition events separated by the appropriate distances, the ratio of transposase complexes to sample DNA is critical. Because the fragment size is also dependent on the reaction efficiency, all reaction parameters, such as temperatures and reaction time, are critical and must be tightly controlled.
Sequencing the genomes of single cells has been recently reported by several group (11, 23-26). The current strategy utilizes whole genome amplification with multiple displacement amplification (MDA). MDA is based on the use of random primers with phi29, a highly processive strand displacing polymerase (27). While this technique is capable of generating enough amplified material to construct sequencing libraries, it suffers from considerable bias, created by nonlinear amplification. A recent report demonstrated a significantly improved method of MDA by adding a quasilinear preamplification step that reduced bias (10). A technology platform based on small compartmentalization and microfluidics can be used to facilitate library preparation from up to 96 single cells per run is offered by Fluidigm (South San Francisco, CA).NGS library construction using RNA
It is important to consider the primary objective of an RNA sequencing experiment before making a decision on the best library protocol. If the objective is discovery of complex and global transcriptional events, the library should capture the entire transcriptome, including coding, noncoding, anti-sense and intergenic RNAs, with as much integrity as possible. However, in many cases the objective is to study only the coding mRNA transcripts that are translated into the proteins. Yet another objective might be to profile only small RNAs, most commonly miRNA, but also small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), and transfer RNA (tRNA). While we will endeavor to describe the principles of RNA sequencing libraries in this review, it is not possible to explain all of the different protocols available. Interested readers should research the many options (Table 1) themselves.
One of the first and earliest successes in applying NGS to RNA-seq was in the case of miRNA (28, 29). The protocols for preparing miRNA sequencing libraries are surprisingly simple and are usually performed in a one-pot reaction (Figure 3). The fact that miRNAs are found in their native state with a 5′ terminal phosphate allows the use of ligases to selectively target miRNAs.
In the first step of the Illumina protocol (Figure 3A), an adenylated DNA adapter with a blocked 3′ end is ligated to the RNA sample using a truncated T4 RNA ligase 2. This enzyme is modified to require the 3′ adapter substrate to be adenylated. The result is that fragments of other RNA species in the total RNA sample are not ligated together in this reaction; only the pre-adenylated oligonucleotide can be ligated to free 3′ RNA ends. Moreover, since the adapter is 3′ blocked, it cannot serve as a substrate for self-ligation. In the next step, a 5′ RNA adapter is added along with ATP and RNA ligase 1. Only RNA molecules whose 5′ ends are phosphorylated will be effective substrates for the ligation reaction. After this second ligation, a reverse transcription (RT) primer is hybridized to the 3′ adapter and a RT-PCR amplification is performed (usually 12 cycles). Due to the small but predictable size of the miRNA library (120 bases of adapter sequence plus the miRNA insert of ~20–30 bases), the library or a pooled sample composed of multiple barcoded libraries can be run on a gel and size selected. The gel size selection is critical due to the presence of adapter dimer side products created during the ligation reaction as well as higher molecular weight products generated from ligation of other non-miRNA RNA fragments containing 5′ phosphate groups (e.g., tRNA and snoRNA). This library preparation method results in an oriented library such that the sequencing always reads from the 5′ end to the 3′ end of the original RNA species. The principle of miRNA sequencing on the Ion Torrent platform is similar (Figure 3B). Ion Torrent uses dual duplex adapters that ligate to the miRNA's 3′ and 5′ ends in a single reaction, followed by RT-PCR. This general library prep approach can also be used to create a directional RNA-seq library from any RNA substrate.