**3.1. DNA and RNA library preparations for second-generation sequencing**

The general workflow for second-generation sequencing is the preparation and amplification of libraries prepared from DNA or RNA samples, clonal formation, sequencing, and analysis [55–59]. Head et al. [55] have reviewed the methods and problems encountered for preparing NGS libraries for whole-genome sequencing, exome sequencing, target sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation sequencing (methyl-seq). Prior to library preparation, the genomic DNA is fragmented by acoustic shearing, sonication, or enzymatic digestion with DNase I or fragmentase and then labeled with adapters, tags, barcodes, and primers using established ligation and PCR methods. Alternatively, Illumina's fragmentation technology, called Nextera Tagmentation, can be implemented using a transposase enzyme to simultane‐ ously fragment and insert adapter sequences into the ds DNA and thereby reduce sample handling and preparation time [57]. For targeted sequencing, the exomes or regions of interest within the fragmented DNA can be captured and enriched by probe-hybridization-capture kits or by PCR amplification with custom-designed primers. For RNA-seq of mRNA, polyA-RNA is isolated usually from total RNA or rRNA-depleted RNA and reverse transcribed to cDNA with reverse transcriptase and polyT or polyU primers before being treated much the same way as the fragmented genomic DNA. RNA sequencing libraries also can be created from immunoprecipitated RNA-binding proteins. To isolate noncoding RNAs (micro, small, and long) from total RNA, these sequences are selectively ligated to 3′ and 5′ adapters and reverse transcribed to cDNA. For methylation sequencing, the genomic DNA is reacted usually with bisulfite chemicals prior to library construction. On the other hand, ChIP-seq and RIPseq use antibody capture to enrich the relevant sequences before preparation of the genomic DNA fragments for sequencing. In comparison to high-input gDNA libraries, the RNA and ChIP libraries may be limited by low cell numbers as starting material and consequently result in a low input of extracted DNA from the immunoprecipitated histones or DNA-binding proteins and in a limited sequence coverage.

Numerous DNA and RNA library kits and machines are available for the semiautomated or fully automated preparation of DNA libraries both for second- and/or third-generation sequencing. Some of these are GemCode from x10 (http://10xgenomics.com) and Raindrop's Thunderstorm (http://raindancetech.com) for all sequencing platforms, cBot for the Illumina platform [58], and Ion Chef and Ion OneTouch for the Ion Torrent platform [59]. All of these kits and auxiliary machines attempt to reduce workload and costs for the main platform sequencers. The DNA libraries are labeled with barcode sample tags, such as the multiplex identifier (MID) for Roche/454 sequencing, to enable the libraries to be pooled and therefore maximize the sequence output as a multiplex amplicon sequencing step for each sequencing run. After library construction, the DNA fragments are clonally amplified by emulsion PCR with microbeads [4, 6, 60] or by solid-phase PCR using primers attached to a solid surface [4, 61, 62] in order to generate sufficient single-stranded DNA molecules and detectable signal for producing sufficiently reliable sequencing data [54]. Roche 454, Life Technologies' SOLiD, and Ion Torrent platforms use emulsion PCR, whereas Illumina's HiSeq/MiSeq platforms use solid-phase PCR [4]. More recently, isothermal PCR amplification on a solid surface of a flow cell [62] was developed for the SOLiD 5500 W series of sequencing machines.

A problem with preparing sequencing libraries by PCR amplification is that PCR introduces GC bias, a major source of unwanted variation and errors in the sequencing coverage [63]. Using alternative methods to PCR amplification improves library complexity and the coverage of high GC regions and reduces the number of duplicate reads [64]. A number of different PCR-free library preparation kits are available commercially, such as NEXTflex PCR-Free from Bioo Scientific, Accel NGS 2S PCR-free library kit from Swift Biosciences, and the Illumina TruSeq DNA PCR-Free Sample Preparation Kit that uses ligation amplification for Illumina and other sequencing platform systems.
