3.2.1. Illumina

Illumina, Inc. [29] is a leading manufacturer of various sequencing instruments. It was established in 1998 in San Diego, CA. Currently, it provides a number of sequencing platforms categorized in two groups: Benchtop Sequencers (BTS) and Production Scale Sequencers (PSS). All BTSs provide support for (1) WGS for small organisms such as microbes and viruses, (2) target gene sequencing (TGS), (3) target gene expression profiling (TGEP), (4) miRNA and sRNA analysis profiling, and (5) 16S metagenomic sequencing (MS) (except iSeq100). NextSeq 550 Series and NextSeq 1000 & 2000 have extra applications such as exome sequencing (ES), s-cell profiling, chip-seq analysis, methylation sequencing, MS, and cell-free sequencing (CFS). Comparison of BTS is given in Table 1. Among PSSs, only NovaSeq 6000 supports WGS of humans, plants, and animals. Functionalities provided by other PSSs are similar to those of benchtop sequencers. Table 2 provides a summary of applications, features, and performance of the PSSs. HiSeq 2500, HiSeq 3000, HiSeq 4000, HiSeq X Ten, and HiSeq X five have been declared to discontinue (Illumina, 2021). However, their support will be provided up to March 31, 2024. So, these sequencers are not discussed here. Illumina sequencing method is based on SBS. Reaction system is a mixture of DNA polymerase, primers, and 4 dNTP with base specific fluorescent markers. The 3′-OH of dNTPs ensures addition of one base at time. On completion of the sequencing reaction, DNA polymerase and the unused dNTP are eluted. For fluorescence excitation, buffer solution is then added. The fluorescence signal is recorded by optical equipment. Optical signals, generated by optical equipment, are used for base calling. To perform next round of sequencing reaction, a chemical reagent is used for quenching fluorescence signal and remove the dNTP 3′-OH protective group. Sequencing data generated during the same experiment have the same length. The latest sequencing platforms can generate DNA sequence in paired-end fashion (22 × 300 bp), i.e., can read both ends of a fragment [30]. Signal decay and dephasing occurred due to incorrect cleavage of fluorescent label or terminating moieties. Average error rates of the sequencing platforms are 1-1.5% [31].

A comparison of Illumina benchtop sequencers [29].

A comparison of Illumina production scale sequencer sequencers [29].