Overview - A multiplatform approach for sequencing
A multiplatform approach in next-generation sequencing (NGS) involves the combined analysis of sequencing reads generated on two or more different sequencing platforms. Most often researchers that require very long read lengths with very low error rates opt for this sequencing strategy. Having physical access to a range of sequencing instruments and the technical knowledge to handle various sequencing technologies are both key elements in properly applying a hybrid sequencing approach.
One successful example of merging sequencing reads from different sequencing systems is the combination of PacBio’s long reads with Illumina’s low-error shorter reads. This cross-platform method is popular for finishing phage, prokaryotic and eukaryotic whole genomes, for resolving complex genomic regions, for phasing haplotypes and for characterising transcripts with high sequence identity.
Applications - The advantages of using cross-platform sequencing
A hybrid approach to next-generation sequencing can lead to:
- Improved error correction
- Decreased number of misassemblies
- Higher number of correctly mapped genomic regions
- Complete genome assembly
- Analysis of full-length cDNA sequences
- Genotyping with increased accuracy
- Identification of novel conformation changes between cancer cells
Sequencing technologies and instruments
Eurofins Genomics houses all state-of-the-art sequencing platforms in its own laboratories. The industry-leading Illumina instruments and the popular PacBio platform can be used alone or in combination to meet the sequencing requirements of a wide variety of projects.
Next-generation sequencing technologies
Eurofins Genomics started out providing NGS services based on the pyrosequencing technology of Roche’s sequencing systems. Pyrosequencing relies on chemiluminescence detection of pyrophosphates that are released during DNA synthesis. The library DNAs with specific adaptors are denatured into single strands and enriched by amplification beads with emulsion PCR. During subsequent DNA synthesis, whenever the DNA polymerase incorporates a nucleotide that is complementary to the DNA template, a pyrophosphate molecule is released and converted to ATP. The ATP molecule provides energy for an enzymatic reaction that generates light. Because nucleotides are added in a stepwise process and their identities are known, the sequence of the template can be determined.
Eurofins Genomics then quickly mastered another sequencing-by-synthesis (SBS) technology that was initially developed by Solexa and later by Illumina. The method involves fixing a library with adaptors, denaturing to single strands and bridge amplifying to form clusters of DNA fragments. The library is then spliced into single strands and sequenced. During sequencing, each of the four kinds of nucleotides that have been modified to contain a cleavable fluorescent dye and a removable blocking group are added sequentially. Upon nucleotide incorporation, a fluorescent signal is emitted and recorded in order to determine the DNA template sequence.
Since then, Eurofins Genomics has also incorporated a third-generation sequencing approach, the single-molecule real-time (SMRT) technology. This sequencing process uses SMRT cells embedded with millions of zero-mode waveguides (ZMWs). A single DNA polymerase is attached to the bottom of a ZMW with a single DNA molecule serving as a template. The structure of the ZMW creates an observation volume that is so small that it only allows the detection of a single nucleotide incorporation event. For the sequencing reaction, four nucleotides are used, each labelled with a different fluorescent dye. When a base is incorporated by the polymerase, the fluorescent tag is cleaved off and the emission signal is recorded in real time. The time difference between two incorporation events can provide additional information about the structural variance and epigenetic modifications of the sequence.
Next-generation sequencing systems
Several high-throughput NGS platforms using corresponding sequencing technologies exist. The most popular sequencing machines include Illumina’s MiSeqs, HiSeqs, and NovaSeq. For long read lengths, the PacBio platforms, which are based on SMRT technology, are the systems of choice.
Products related to multiplatform sequencing
Are you convinced that a cross-platform approach will benefit your next-generation sequencing project? Try our NGSelect products NGSelect DNA, NGSelect RNA, NGSelect Amplicon and NGSelect Ready-to-Load for maximum flexibility in the generation and analysis of sequencing reads.
Are you interested in seeing how Illumina’s platforms can advance your research? Rely on one of our INVIEW pre-designed services - NGS Built For You –
Ready to explore with PacBio’s ultra-long reads? See how you can use one of our standardised INVIEW products to enhance your de novo genome finishing with the SMRT technology. Take advantage of our all-in-one packages offering DNA isolation, library preparation, sequencing and BioIT analysis all performed by a team of multi-disciplinary experts.
Please contact us with any questions on how our sequencing platforms can be used to achieve your project objectives.