Illumina Sequencing
Illumina Sequencing - Related Videos
14:49
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
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Cited by 30 •
2011
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
08:57
Amplicon Sequencing using the Long-Read Sequencing Technologies
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2025
This protocol was optimized for targeted deep sequencing of 18 drug-resistance regions in Mycobacterium tuberculosis using a long-read sequencing platform, followed by analysis with a tuberculosis-specific bioinformatics pipeline designed for long-read data.
07:30
Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples
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Cited by 19 •
2020
This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.
02:02
Cis-regulatory Sequences
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2020
Cis-regulatory sequences are short fragments of non-coding DNA that are present on the same chromosomes as the genes that they regulate. These fragments serve as binding sites for transcriptional regulators, proteins that are responsible for controlling gene transcription and differential gene expression across cell types in eukaryotes. Cis-regulatory sequences can be close to the gene of interest or thousands of bases away in the DNA sequence; however, those sequences that are further away are...
04:09
High-Resolution Melting PCR to Determine Sequence Variations in Mutant DNA Sequences
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2025
In this video, we describe a high-resolution melting PCR technique to determine the length polymorphism of DNA fragments of varying sequence lengths, based on melting temperature analysis.
FAQs
Bridge amplification creates a clonal cluster from each immobilized DNA fragment on the flow cell. The resulting group contains many copies of the same starting fragment rather than a single molecule, allowing fluorescent incorporation events to be imaged during sequencing. This amplification supports analysis of many DNA fragments in parallel while preserving the sequence identity of each cluster.
Each fluorescently labeled, reversible-terminator nucleotide permits one base to be added at a time. Imaging records the fluorescence associated with that incorporation, identifying the base in the growing strand. The terminator is then reversible, allowing the next cycle to proceed. Repeating this controlled process produces successive base calls for each DNA fragment.
The instrument records fluorescence after each nucleotide-incorporation cycle, and each signal identifies the base added to a fragment. Computational analysis then assembles these sequential base calls into the corresponding DNA sequence. This combination of repeated imaging and computational reconstruction enables detailed analysis while supporting the technology’s accuracy and high-throughput operation.
The workflow first places DNA fragments on a flow cell, where bridge amplification generates a clonal cluster from each fragment. Sequencing by synthesis then adds fluorescently labeled, reversible-terminator nucleotides through repeated cycles. Imaging identifies every incorporated base, and computational analysis reconstructs the sequences for downstream biological interpretation.
Illumina sequencing can support genome analysis, transcriptome analysis, gene expression profiling, variant detection, metagenomics, and biomedical research. The same underlying data-generation approach can therefore be applied to questions about genetic composition, expressed genetic material, sequence differences, or the mixture of organisms present in a biological sample.
Its accuracy, scalability, and relatively low cost make Illumina sequencing practical for analyzing large numbers of DNA fragments and diverse biological samples. In biology, the resulting sequence information can help characterize genomes and transcriptomes, detect variants, examine gene expression, study microbial communities through metagenomics, and support broader biomedical investigations.