NGS library construction(未完待续)

基本原理：Fundamental to NGS library construction is the preparation of the nucleic acid target, RNA or DNA, into a form that is compatible with the sequencing system to be used (Figure 1).

Figure 1.  Basic workflow for NGS library preparation. RNA or DNA is extracted from sample tissue/cells and fragmented. RNA is converted to cDNA by reverse transcription. DNA Fragments are converted into the library by ligation to sequencing adapters containing specific sequences designed to interact with the NGS platform, either the surface of the flow-cell (Illumina) or beads (Ion Torrent). The next step involves clonal amplification of the library, by either cluster generation for Illumina or microemulsion PCR for Ion Torrent. The final step generates the actual sequence via the chemistries for each technology. One difference between the two technologies is that Illumina allows sequencing from both ends of the library insert (i.e., paired end sequencing). Cell photograph courtesy of Annie Cavanagh, Wellcome Images.

需要注意的一些方面：The size of the target DNA fragments in the final library is a key parameter for NGS library construction.

核酸链片段化方法：Three approaches are available to fragment nucleic acid chains: physical, enzymatic, and chemical. DNA fragmentation is typically done by physical methods (i.e., acoustic shearing and sonication) or enzymatic methods (i.e., non-specific endonuclease cocktails and transposase tagmentation reactions)

上述方法的实际效果：In our laboratory, acoustic shearing with a Covaris instrument (Covaris, Woburn, MA) is typically done to obtain DNA fragments in the 100–5000 bp range, while Covaris g-TUBEs are employed for the 6–20 Kbp range necessary for mate-pair libraries.

Enzymatic methods include digestion by DNase I or Fragmentase, a two enzyme mix (New England Biolabs, Ipswich MA). Comparisons of NGS libraries constructed with acoustic shearing/sonication versus Fragmentase found both to be effective. However, Fragmentase produced a greater number of artifactual indels compared with the physical methods. An alternative enzymatic method for fragmenting DNA is Illumina's Nextera tagmentation technology (Illumina, San Diego, CA) in which a transposase enzyme simultaneously fragments and inserts adapter sequences into dsDNA. This method has several advantages, including reduced sample handling and preparation time

文库大小和插入长度的问题：Desired library size is determined by the desired insert size (referring to the library portion between the adapter sequences), because the length of the adaptor sequences is a constant. In turn, optimal insert size is determined by the limitations of the NGS instrumentation and by the specific sequencing application.

例子：For example, when using Illumina technology, optimal insert size is impacted by the process of cluster generation in which libraries are denatured, diluted and distributed on the two-dimensional surface of the flow-cell and then amplified. While shorter products amplify more efficiently than longer products, longer library inserts generate larger, more diffuse clusters than short inserts. We have successfully sequenced libraries with Illumina instruments up to 1500 bases in length.

根据目的选文库size：

Optimal library size is also dictated by the sequencing application. For exome sequencing, more than 80% of human exomes are under 200 bases in length. We run 2 × 100 paired-end reads and our exome sequencing libraries typically contain insert sizes of approximately 250 bases in length as a compromise to match the average size of most exons while sequencing without overlapping read pairs. The size of an RNA-Seq library is also determined by the applications. We typically do basic gene expression analysis using single-end 100 base reads. However, for analysis of alternative splicing or determination of transcription start and stop sites, we employ 2 × 100 base paired-end reads. In most instances, the RNA will be fragmented before conversion into cDNA. This is typically done through the use of controlled heated digestion of the RNA with a divalent metal cation (magnesium or zinc). The desired length of the library insert can be adjusted by increasing or decreasing the time of the digestion reaction with good reproducibility.

RNA-seq 文库制备方法：

seven different RNA-seq library preparation methods , the majority involve some sort of fragmentation of the mRNA prior to adapter attachment. The two that do not use a hexamer priming method or in the case of the SMARTer Ultra Low RNA Kit (Clontech, Mountain View, CA), a full length cDNA is synthesized with a fixed 3′ and 5′ sequence added so that the entire cDNA library (average 2 kb in length) can be amplified in long distance PCR (LD-PCR). This amplified double-stranded cDNA is then fragmented by acoustic shearing to the appropriate size and used in a standard Illumina library preparation (involving end-repair and kination, A-tailing and adapter ligation, followed by additional amplification by PCR).

A second post-library construction sizing step is commonly used to refine library size and remove adaptor dimers or other library preparation artifacts. Adapter dimers are the result of self-ligation of the adapters without a library insert sequence. These dimers form clusters very efficiently and consume valuable space on the flow cell without generating any useful data. Thus, we typically use either magnetic bead-based clean up, or we purify the products on agarose gels. The first works in most instances for samples where sufficient starting material is available. When sample input is limiting, more adapter dimer products are often generated. In our experience, bead-based methods may not perform optimally in this situation and combining bead-based with agarose gel purifications may be necessary.