Illumina uses sequencing by synthesis (SBS) technology and is considered the most successful and widely adopted next-generation sequencing platform worldwide. Illumina sequencing technology has become more and more popular in basic biological and medical research due to its low sequencing errors, high output, and flexible applications. Especially, MiSeq, the industry's most accurate and easiest-to-use benchtop sequencer has been widely used in microbial ecology studies since new MiSeq reagents enable up to 15 Gb of output with 25 M sequencing reads and 2 x 300 bp read lengths. MiSeq is able to access even more applications such as target gene sequencing (TGS), shotgun metagenome sequencing (SMS), and metatranscriptome sequencing (MTS).
Target gene sequencing (TGS)
Community DNA is extracted from environmental samples (e.g., soils, sediments, water, bioreactors, human bodies), purified, quantified and used as templates. Selected target genes, either phylogenetic markers (e.g., 16S rRNA gene), or functional markers (e.g., amoA, nifH, dsrA, nosZ) should have a conserved primer pair, which is combined with a unique barcode/linker and an adapter for synthesizing a fusion primer pair, and this primer pair is then used for PCR amplification for each sample. It is ideal that the length of amplicons is within 550 bp when 2x300 bp read lengths are generated. Obtained PCR amplicons from each sample are purified, and quantified, and then many samples (usually 20-200 samples) are equally pooled, and sequenced by MiSeq. For 16S rRNA-based microbial ecology studies, the gene region of F515-R806 (based on E. coli 16S rRNA gene positions) is most widely used.
Shotgun metagenome sequencing (SMS)
Although targeted gene sequencing is a powerful tool for providing information on specific targets within a microbial community, its suitability for analyzing the whole genetic and functional diversity of communities is limited. To query broader characteristics and identify novel genes, shotgun metagenome sequencing (SMS) has been widely used. Briefly, community DNA is randomly sheared using various methods including nebulization, endonucleases, and ultrasonication. The sheared fragments are end-repaired prior to ligation to platform-specific adaptors, which serve as the priming sites for template amplification. Recently, a transposon-based approach for fragmentation and tagging has become available. Subsequent sequencing produces vast amounts of short reads, which can be assembled and annotated for functional characterization.
Metatranscriptome sequencing (MTS)
Metatranscriptomics involves random sequencing of microbial community mRNA. Typically, total RNA extracted from microbial communities is dominated by rRNA, which must be removed to obtain high levels of mRNA transcripts. Then, the remaining RNAs are reverse transcribed into cDNAs, ligated to adapters and sequenced. Major challenges include the inherent liability of mRNA that requires proper stabilization and storage procedures, efficient rRNA removal prior to reverse transcription, as well as obtaining sufficient quantities of RNA to avoid the need for amplification.