Next-generation Sequencing (NGS)

What Is Next-Generation Sequencing?

Next-generation sequencing (NGS), also called high-throughput sequencing or massively parallel sequencing, determines the nucleotide sequence of millions to billions of DNA or RNA fragments simultaneously in a single instrument run. Unlike Sanger sequencing, which reads one locus per reaction, NGS uses sequencing-by-synthesis (Illumina), nanopore translocation (ONT), or single-molecule real-time (SMRT) chemistry (PacBio) to sequence an entire genome, transcriptome, metagenome, or targeted panel in hours to days. Choose NGS reagents based on your sequencing application (DNA-seq vs. RNA-seq vs. amplicon), target platform (Illumina, ONT, or PacBio), sample type (fresh, FFPE, cfDNA, low-input), and throughput requirements.

What you will find out:

• NGS Library Preparation Kits: Effective options like the Zymo-Seq SwitchFree series and MagQuant Plus DNA kits, tailored to streamline library production while improving molecular diversity and reducing manual effort.

• RNA-sequencing: Customized mRNA and total RNA library kits created for exact gene expression assessments, ensuring precise quantification of low-abundance transcripts with limited starting material.

• DNA-sequencing: Trustworthy genomic DNA extraction methods designed for high-yield retrieval and uniform coverage, providing a strong foundation for whole-genome sequencing (WGS) and targeted resequencing projects.

How to Choose NGS Library Prep Reagents

Application type

DNA sequencing applications include whole-genome sequencing (WGS), whole-exome sequencing (WES), targeted gene panels, amplicon sequencing (16S/ITS for microbiome, custom amplicons), and epigenetic sequencing (WGBS, RRBS, ChIP-seq). RNA sequencing applications include mRNA-seq (poly-A enriched), total RNA-seq (rRNA depleted), small RNA-seq (miRNA, piRNA), and single-cell RNA-seq. Each application requires a distinct library prep chemistry and kit format.

Platform compatibility

Confirm library prep kit compatibility with the target sequencing platform before ordering. Illumina-compatible kits are the most common and generate short-read libraries with paired-end 150 bp reads on NovaSeq X, NextSeq 2000, or MiSeq instruments. ONT-compatible kits generate long-read libraries for MinION, GridION, or PromethION. PacBio HiFi kits generate ultra-accurate long reads on the Revio or Sequel IIe. Most Zymo Research NGS library prep kits are optimized for Illumina sequencing.

Sample type and input amount

Standard input amounts are 100 ng-1 microgram of gDNA for WGS, 1-100 ng for targeted panels, and 10-100 ng of total RNA for RNA-seq. Low-input kits support 1-100 pg of total RNA (single-cell or ultra-low input). FFPE-compatible kits use specialized fragmentation and repair chemistry to address formalin-induced crosslinks and DNA damage, accepting inputs as low as 10-50 ng of FFPE-extracted DNA. Confirm the minimum input requirement and the DV200 score threshold for FFPE RNA before selecting a kit.

Zymo Research NGS portfolio at MBP

Zymo Research is a key MBP brand with an NGS library prep portfolio spanning: Zymo-Seq RiboFree Total RNA Library Kit (probe-free rRNA depletion, any organism, 10 ng minimum RNA input, cited in 150+ peer-reviewed studies); Quick-16S Plus NGS Library Prep Kits (normalization-free, 30 minutes hands-on time for 96 samples, V1-V2 or V4 16S rRNA region); Zymo-Seq RRBS Library Kit (reduced representation bisulfite sequencing, as little as 2 hours hands-on time); and Zymo-Seq UDI Primer Sets (unique dual index barcodes for multiplexed sequencing). Zymo Research kits are cited extensively in published microbiome, transcriptomics, and epigenomics research.

Automation compatibility

High-throughput genomics cores and biobanks processing hundreds of samples per week benefit from automation-compatible library prep formats. Zymo Research Quick-16S Plus kits are 100% automation ready and require no normalization step between PCR and pooling. Assess liquid-handling robot compatibility (Hamilton, Beckman, Tecan, Agilent Bravo) with the kit vendor before scaling.

Specifications Context

NGS library quality is assessed by three metrics: concentration (Qubit fluorometry; target 2-10 nM for Illumina clustering), fragment size distribution (Bioanalyzer or TapeStation; target 300-500 bp insert for standard Illumina), and library complexity (assessed by PhiX spike-in error rate and percentage of duplicate reads). Adapter dimer contamination below 5% of total library molecules is recommended before sequencing; size selection with SPRI beads (such as AMPure XP, 0.8-1.0X bead ratio) removes dimers efficiently.  PCR-free WGS library preparation is increasingly preferred for high-coverage sequencing to eliminate PCR amplification bias, particularly in GC-rich and AT-rich regions, and requires a minimum input of 200-500 ng of high-molecular-weight DNA (HMW gDNA). 

Boost your genomic knowledge—contact the MBP team today for a quote on our specialized NGS preparation services.