Klenow fragment ligation9/13/2023 ![]() In most applications, this large fraction is irrelevant to the question of interest. Ribosomal RNA (rRNA) makes up more than 80–90% of the total RNA pool of all cells ( 24–26). Nevertheless, the downstream steps for most of these molecules are generally the same. For each of these subsets, dedicated protocols ( 17–23) or commercial kits exist for their purification-these are beyond the scope of this review and will not be detailed further. The starting point in RNA procedures are mostly total RNA or poly(A) +-RNA transcripts, but can extend to in vitro-transcribed (IVT) RNA, various types of non-coding RNAs, ribosome footprints, tRNAs, crosslinked RNA or modified RNA. The plethora of different types of libraries all converge to dealing with either DNA or RNA (which is, eventually, almost always converted into amplifiable DNA). ![]() We here summarized the principal insights in this fast-paced discipline, expanding on newly published studies and aspects not covered in previous reviews ( 14–16). The goal of this review is to provide an in-depth yet application-independent overview of current and state-of-the-art technical developments in the field, guiding the reader through the vast expanse of tools that can be used to turn a pool of nucleic acids into a library that can be sequenced or assayed using other means. Additionally, there is always a lag between the description of a new method and its commercialization. transcriptome sequencing), it is generally acknowledged that these conventional procedures allow little room to tailor the library toward the specific needs of the researcher, especially when the research question calls for a non-standard approach. While there are numerous suppliers for sequencing library construction, and the resulting libraries are often of reasonable quality for standard sequencing experiments (e.g. Many researchers can (and do) resort to the use of commercial kits to capture the desired nucleic acid species into a workable library of molecules. Coding sequence fragment libraries are a prominent example ( 10–13). In several other cases, however, such as for very large libraries or libraries with custom requirements, high-quality libraries still need to be generated. Fortunately, some of these libraries can now be accurately synthesized at relatively low cost, or one can rely on available collections of full-length and validated open reading frames (ORFs) on plasmids ( 7), short hairpin or small interfering RNA libraries ( 8) and guide RNA libraries for CRISPR screens ( 9). In addition to libraries for sequencing purposes, many proteome-wide functional assays, for instance assessing protein interactions ( 2, 3), protein localization ( 4), post-transcriptional regulation ( 5) or drug activity ( 6), also rely on pooled or arrayed nucleic acid libraries as input. Indeed, in the past few years, the number of studies reporting (and in many, cases, addressing) the impact of the choice of specific enzymes, reagents, reaction conditions or overall protocols on the resulting library quality have grown exponentially, and there is renewed interest in the development of molecular biology tools designed to overcome these biases. Additionally, a higher quality library usually maximizes the useful sequencing read output and facilitates data processing. The paramount role of library construction is often underappreciated, yet it shapes both outcome and inference: the library protocol should meticulously capture the specific molecules of interest, yet minimize unwanted fragments or biases in order to ensure accurate interpretation (‘garbage in is garbage out’). The fast-paced methodological progress driving many of the developments in the field has not only been the result of exceptional advances in sequencing chemistry, detection systems and data-processing or analysis methods ( 1), but also of innovations in the area of sequencing library construction. Next generation sequencing technologies have undeniably changed the scientific landscape in biology.
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