High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

Many candidate unnatural DNA base pairs have been developed, but surprisingly, some of the best replicated adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replicating one of the more promising pairs, dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.

Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to the identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of E. coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivatives are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM and d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the minimum overall fidelity ranges from 10 3 to 10 4 . In addition, the data allow us to propose a generalized model of unnatural base pair replication, which should aid in the further optimization of the unnatural base pair, and possibly in the design of additional unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.

We synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addition, the dTPT3 scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3PA-dNaM pair, which is amplified only slightly less well. The identification of dTPT3 represents significant progress towards developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnology applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.

Expansion of the genetic alphabet with a third base pair would lay the foundation of a semi-synthetic organism with an expanded genetic code and also have immediate in vitro applications. Previously, the unnatural base pairs formed between d5SICS and either dNaM or dMMO2 were shown to be well replicated by DNA polymerases under steady-state conditions and also transcribed by T7 RNA polymerase efficiently in either direction. We now demonstrate that DNA containing either the d5SICS-dNaM or d5SICS-dMMO2 unnatural base pair may be PCR amplified with fidelities and efficiencies that approach those of fully natural DNA. These results further demonstrate that the determinants of a functional unnatural base pair may be designed into predominantly hydrophobic nucleobases with no structural similarity to the natural purines or pyrimidines. Importantly, the results reveal that the unnatural base pairs may function within an expanded genetic alphabet and make possible many in vitro applications.The genetic alphabet is constrained by the efficient polymerase-mediated replication of DNA and RNA containing the two natural base pairs. In addition to laying the foundation for a semisynthetic organism with an expanded genetic code, an efficiently and selectively replicated and transcribed unnatural base pair would dramatically increase the potential of the already ubiquitous in vitro methodologies based on DNA and RNA, and their sequence specific amplification. PCR amplification of DNA containing unnatural base pairs was first reported by Benner and co-workers, using pairs with orthogonal H-bonding complementarity, 1 followed by Hirao and co-workers, 2 using a pair formed between substituted pyrimidine and pyrrole nucleotide analogs. These studies represent landmarks in the effort to expand the genetic alphabet, but the unnatural pairs used are limited by strong sequence dependencies and/or inefficient transcription into RNA. This may not limit their use in some in vitro applications, but does reduce their generality and preclude their eventual use in vivo as part of a semisynthetic organism.We have focused on developing unnatural base pairs formed between predominantly hydrophobic nucleobases that have no structural homology to the natural nucleobases, and that pair based on hydrophobic and packing forces. Screening of a library of nucleotides, followed by hit optimization, identified the pair formed between d5SICS and dMMO2 (Figure 1) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript nucleotide incorporation conditions. 3a Further optimization identified dNaM, which pairs with d5SICS to form an unnatural pair that is even better replicated under the same steady state conditions. 3b Importantly, d5SICS-dMMO2, and especially d5SICS-dNaM, are also efficiently transcribed in both directions, 3c suggesting that they might have immediate practical applications. Here, we examine whether these unnatural base pairs, which are much less natural-like than those previously examined, are also sufficiently w...