One major limitation in miRNA library construction arises when the amount of input RNA is low (e.g., <200 ng total RNA); short adapter dimers compete in the RT-PCR reaction with the desired product, adapters, and miRNA inserts. When too many adapter dimers are present they stream up the gel during the size selection step and contaminate the product bands. To minimize this problem, many commercial miRNA library preparation kits now incorporate various strategies to suppress adapter dimer formation.
For mRNA sequencing libraries, methods have been developed based on cDNA synthesis using random primers, oligo-dT primers, or by attaching adapters to mRNA fragments followed by some form of amplification. mRNA can be primed by random oligomers or by an anchored oligo-dT to generate first strand cDNA. If random priming is used, the rRNA must first be removed or reduced. rRNA can be removed using oligonucleotide probe-based reagents, such as Ribo-Zero (Epicenter, Madison, WI) and RiboMinus (Life Technologies, Carlsbad, CA). Alternatively, poly-adenylated RNA can be positively selected using oligo-dT beads.
It is often desirable to create libraries that retain the strand orientation of the original RNA targets. For example, in some cases transcription creates anti-sense RNA constructs that may play a role in regulating gene expression (30). In fact, long noncoding RNA (lncRNA) analysis depends on directional RNA sequencing (31). Methods for preparing directional RNA-seq libraries are now readily available (15). The concept is to perform the cDNA reaction and remove one of the two strands selectively, by incorporating dUTP into the second strand cDNA synthesis reaction. The uracil-containing strand can then be removed enzymatically (32) (NEBNext Ultra Directional RNA Library Prep Kit for Illumina) or prevented from further amplification with a PCR polymerase that cannot recognize uracil in the template strand (Illumina TruSeq Stranded Total RNA kit). In addition, actinomycin D is frequently added to the first strand cDNA synthesis reaction to reduce spurious antisense synthesis during the first strand synthesis reaction (33).
An alternative and hybrid method utilizes random or anchored oligo-dT primers with an adapter sequence on the 5′ end of the primer to initiate first strand cDNA synthesis. Next, in a procedure called template switching (shown in Figure 4B), a 3′ adapter sequence is added to the cDNA molecule (17). This method has a distinct advantage in that the first strand cDNA molecule can be PCR amplified directly without second strand synthesis using the unique sequence tag put on the 3′ end by the template switching reaction. A 5′ unique sequence tag is also introduced by standard priming in the first strand synthesis.
The strategic design of the primers used for cDNA synthesis is a powerful strategy for making RNA-seq libraries. For example, rRNA sequences can be avoided by including strategically designed primers that target rRNA but do not allow subsequent amplification. A commercial kit (NuGEN Ovation RNA-seq; San Carlos, CA) combines SPIA nucleic acid amplification technology (34) with primers used in the first strand cDNA synthesis that are designed to suppress amplification of rRNA sequences. Another method was reported in which all 4096 possible hexamer sequences were screened against rRNA sequences to identify and eliminate perfect matches. A pool of 749 hexamers remained that was then used to prime the first strand cDNA synthesis reaction. The result was a drop in rRNA reads from 78% to 13% in the sequencing data (16). Finally, a method called DP-seq (7) was developed, in which the amplification of a majority of the mouse transcriptome was accomplished using a defined set of 44 heptamer primers. This primer sequence design selectively suppressed the amplification of highly expressed transcripts, including rRNA, and provided a reliable estimation of low abundance transcripts in a model of embryonic development.
Recently methods for preparing RNA-seq libraries from single cells have been reported (Figure 4)(3-5, 8, 9). One strategy utilizes polynucleotide tailing of the first strand cDNA (Figure 4A)(5, 8), which can be combined with a template switching reaction (Figure 4B)(4, 9). The end result is a first strand cDNA product that can be amplified by universal PCR primers. The version shown in Figure 4B has been incorporated into a commercially available kit (SMARTer Ultra Low RNA Kit; Clontech). An alternative approach called CEL-Seq incorporates a T7 promoter sequence at the 5′ end of the cDNA, followed by linear amplification using in vitro transcription (Figure 4C)(3).