Since a missing 3′ T would lead to adapter dimer formation, the more stable phosphorothioate linkage (instead of phosphodiester) is usually used to attach the 3′ T to the adapters. Additional T on the 3′ end of the P5 adapter to prevent formation of adapter dimers and facilitate ligation with the 3′ A of library fragments (similar to TA cloning).Index sequences enable multiplexing, a process of sequencing multiple libraries in one flow cell, and dual-indexed libraries are commonly employed for multiplex sequencing ( Figure 13). Index sequences composed of specific 6–8 nucleotides to distinguish one sample from another.Sites of binding to P5 or P7 oligos on the flow cells and to the sequencing primers.This Y shape is no longer maintained if library amplification is subsequently performed ( Figure 11). The adapters are noncomplementary at their ends to prevent their self-ligation and thus form a Y shape after annealing. The P5 and P7 adapters are named after their sites of binding to the flow cell oligos. Because library fragments are flanked by adapters, they are sometimes called inserts.ĭuring formation of the adapter duplexes, two strands of oligos called P5 and P7 are annealed. Ligation efficiency is critical for conversion of DNA fragments into sequenceable molecules and thus impacts conversion rate and yield of the libraries. In library preparation, a stoichiometric excess of adapters relative to sample DNA is used to help drive the ligation reaction to completion. Identical duplex adapters are ligated to both ends of the library fragments so that oligos on the flow cell can recognize them for sequencing. Resting/cooling periods between sonication cycles should be incorporated to keep the samples from overheating, which necessitates a longer workflow and wait time.Īdapters are a pair of annealed oligonucleotides that facilitate clonal amplification and sequencing reactions. In either approach, optimization is needed to obtain the desired fragment lengths ( Figure 8). Waterbath-based sonication, on the other hand, keeps the samples within a closed system but usually requires higher energy due to energy dissipation/dispersion and low output. Although probe-based sonication delivers more focused energy towards the sample, the samples are in an open container, directly in contact with the probe, and thus are at a high risk of contamination. Sonication is the simplest method among the three and uses a sonicator (probe- or waterbath-based) to emit low-frequency acoustic waves for shearing.Widely used methods include high-power unfocused sonication, nebulization, and focused high-frequency acoustic shearing. Mechanical shearing involves breakage of phosphodiester linkages of DNA molecules by applying shear force. Target enrichment by hybrid capture usually requires higher sample input and a longer workflow, but it may yield more uniform coverage and higher data quality over the PCR enrichment method. After removing the unbound sequences, target sequences are released from the probes and prepared as a sequencing library ( Figure 5). Probes are usually coupled to magnetic or biotinylated beads so that target sequences hybridized to the probes can be selected from the mixture. The hybrid capture strategy utilizes a set of oligonucleotide probes that are complementary to the target sequences.Among methods available for enrichment of target sequences, the two most common approaches are hybrid capture and PCR amplification.
To perform targeted sequencing, samples are enriched for the sequences of interest. Since targeted sequencing does not require analysis of the whole genome (e.g., 3.2 x 10 9 base pairs for human), it allows more reads, better coverage, and higher depth, and therefore improved detection of rare variants at a lower cost than WGS. Therefore, targeted sequencing is hypothesis-driven and requires knowledge of the sequence of the reference genes or genomic regions. An example of targeted sequencing is screening for known cancer genes in different types of cancer cells. Targeted sequencing (instead of WGS) is used when the goal of the experiment is to sequence specific genes, sets of related genes, or targeted regions of a genome.
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