Animal/Plant Long Amplicon Sequencing

DNA barcoding (COI, rbcL, matK, ITS), transgene verification, targeted genotyping, and haplotype-resolved locus analysis by PacBio HiFi and Oxford Nanopore dual-platform long-read amplicon sequencing — comprehensive species coverage across mammals, birds, fish, insects, plants, and more, not limited to commonly studied organisms

CD Genomics provides long amplicon sequencing for animal and plant research using PacBio HiFi (Q30+ consensus accuracy) and Oxford Nanopore PromethION platforms, supporting PCR amplicons from 1 kb to 15+ kb. Our service covers full-length DNA barcoding (COI, rbcL, matK, ITS), transgene insertion verification, targeted genotyping, and haplotype-resolved locus analysis across any species — including non-model organisms without reference genomes.

Long amplicon sequencing bridges the gap between short-read amplicon panels and whole-genome sequencing by delivering kilobase-length, single-molecule reads across targeted genomic regions. At CD Genomics, we combine PacBio HiFi circular consensus sequencing and Oxford Nanopore long-read technologies to provide end-to-end long amplicon sequencing services for animal and plant research. Whether your goal is species identification through DNA barcoding, transgene insertion validation, targeted genotyping of agronomic traits, or haplotype-resolved analysis of multi-kb loci, our dual-platform approach delivers the read length, accuracy, and throughput to match your experimental requirements.

Our service supports PCR amplicons ranging from 1 kb to 10+ kb across the full spectrum of commonly used genetic markers — mitochondrial (COI, Cytb, 12S, 16S, D-loop), chloroplast (rbcL, matK, psbA-trnH), nuclear ribosomal (ITS1, ITS2, full-length ITS, 18S, 28S), and custom-designed target regions — for any animal or plant species, including non-model organisms without existing reference genomes. For full-length 16S and ITS ribosomal amplicon applications, see our Full-Length 16S/18S/ITS Amplicon Sequencing service.

Why Choose Our Long Amplicon Sequencing Service

• Dual-Platform Flexibility — PacBio HiFi (Q30+ consensus accuracy) for high-confidence variant detection and ONT PromethION for cost-effective deep coverage across large amplicon panels
• Broad Species Coverage — Proven protocols for mammals, birds, reptiles, fish, insects, mollusks, crop plants, trees, ornamental species, and non-model organisms — not limited to standard laboratory species
• kb-to-Mb Scalable Amplicon Panels — From single-locus barcoding to multiplexed panels of 50+ targets in a single run, leveraging PacBio multiplexing with 384 barcodes and ONT native barcoding
• End-to-End Bioinformatics — Dedicated analysis pipelines for CCS/HiFi read processing, demultiplexing, consensus generation, variant calling (SNPs, InDels, STRs), haplotype phasing, and species-level taxonomic assignment
• Field-to-Sequence Flexibility — Accepts extracted gDNA, tissue, blood, FTA cards, and environmental samples; custom PCR optimization included for challenging templates
• Rapid Turnaround — Standard project completion in 15–25 business days with real-time progress tracking and intermediate QC checkpoints

Long Amplicon Sequencing Spans Complete Genes and Haplotypes That Short-Read Amplicon Methods Cannot Resolve

Long amplicon sequencing refers to the targeted PCR amplification and high-throughput sequencing of genomic regions ranging from approximately 1 kb to 15+ kb in length, using long-read sequencing platforms capable of spanning entire genes, multigenic clusters, or repetitive regions in single contiguous reads. Unlike short-read amplicon sequencing (e.g., Illumina MiSeq 2×300 bp), which requires fragmenting target regions into overlapping tiles and computationally reconstructing full-length sequences, long-read amplicon sequencing reads each template molecule from end to end in a single pass, preserving complete gene structure, phasing information, and linkage between distant variants.

For animal and plant research, this technical distinction is transformative. A full-length COI barcode (658 bp), the standard mitochondrial marker for metazoan species identification, can be sequenced in a single PacBio HiFi read at Q30+ accuracy — eliminating the need for tiled amplicons, reducing PCR bias, and enabling direct observation of heterozygous haplotypes. Similarly, the complete ITS1-5.8S-ITS2 ribosomal cassette (~650–1,200 bp in plants) or the full 16S-ITS-23S ribosomal operon (~4,500 bp) can be captured as single contiguous amplicons, providing phylogenetic resolution that is unattainable with short-read approaches.

At CD Genomics, we have optimized long amplicon sequencing protocols across both major long-read platforms — PacBio (Sequel II / Revio with HiFi CCS) and Oxford Nanopore (PromethION with R10.4.1 flow cells) — to support animal and plant researchers working with any species of interest. Our service covers the complete workflow from PCR optimization and primer design through library preparation, multiplexed sequencing, and species-aware bioinformatics analysis.

Long Amplicon Sequencing Delivers Full-Length Single-Molecule Resolution with Species-Level Taxonomic Accuracy

Long Amplicon Sequencing Supports DNA Barcoding, Transgene Verification, Genotyping, and Phylogenetic Analysis

Species Identification & DNA Barcoding

• Full-length COI barcoding for animal species identification, including cryptic species discrimination in biodiversity surveys and forensic wildlife tracking. See our Long-Read Metagenomics Sequencing service for eDNA metabarcoding applications
• Multi-locus plant barcoding (rbcL + matK + ITS) for botanical authentication, herbarium specimen verification, and invasive species monitoring
• Fungal ITS barcoding for mycological surveys, plant pathogen detection, and industrial strain authentication
• eDNA metabarcoding with full-length barcode genes for non-invasive biodiversity monitoring from soil, water, and air samples

Transgene & Gene Editing Verification

• Full-length sequencing of transgene insertion cassettes to confirm intact integration, copy number, and absence of vector backbone contamination
• CRISPR/Cas9, TALEN, and ZFN editing outcome verification — spanning the edited locus to detect intended edits, off-target mutations, and structural rearrangements. For genome-wide characterization of edited lines, see our Whole Genome De Novo Sequencing service
• Characterization of large genomic deletions, insertions, and allele-specific modifications in transgenic animal and plant lines

Targeted Genotyping & Marker Discovery

• High-resolution genotyping of SSR/STR loci, SNP panels, and agronomic trait-associated haplotypes in breeding populations
• Full-length amplicon sequencing of MHC, immune gene complexes, and disease-resistance gene clusters for association studies
• Population-level screening of mitochondrial and chloroplast haplotypes for germplasm characterization and conservation genetics

Haplotype-Resolved Locus Analysis

• Direct phasing of multi-kb heterozygous regions without statistical inference — single-molecule reads span both haplotypes
• Characterization of structural variants (InDels, CNVs, inversions) within targeted genomic intervals
• Allele-specific expression and imprinting analysis through long amplicon sequencing of cDNA targets

Phylogenetics & Evolutionary Genomics

• Multi-locus phylogenetic reconstruction using concatenated mitochondrial, chloroplast, and nuclear ribosomal markers
• Population genomics and phylogeography through targeted resequencing of variable regions across populations
• Molecular clock calibration and divergence time estimation using full-length gene sequences from multiple taxa

Our Bioinformatics Pipelines Deliver Consensus Sequences, Variant Calls, and Taxonomic Assignment from Long Amplicon Reads

Our bioinformatics pipelines are tailored specifically for long-read amplicon data and cover all standard deliverable types required for publication. The table below details our analysis modules across basic and advanced tiers.

Sample Input and Quality Requirements for Long Amplicon Sequencing

Case Study: Nanopore-Based Species Identification of Neotropical Non-Model Mammals

Velasquez-Restrepo S, Corrales Orozco M, Franco-Sierra ND, Martínez-Cerón JM, Díaz-Nieto JF. Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore. PeerJ. 2024;12:e17887. doi:10.7717/peerj.17887.

1. Background

Species identification is a critical first step in biodiversity research, conservation biology, and ecological monitoring. Traditional morphological identification requires specialized taxonomic expertise and is often insufficient for cryptic species complexes. While DNA barcoding (primarily COI) offers a molecular alternative, the conventional barcoding workflow relies on Sanger sequencing of individual specimens — a low-throughput and costly approach that becomes impractical at the scale required for biodiversity inventories in megadiverse regions such as the Neotropics.

In this study, Velasquez-Restrepo et al. set out to develop and validate a Nanopore-only workflow for rapid species identification of non-model small mammal species (rodents, bats, and marsupials) from field-collected samples in Colombia's northern Cordillera Central, using Oxford Nanopore MinION sequencing and a custom bioinformatics pipeline for mitochondrial genome reconstruction from shotgun data.

2. Methods

Field sampling was conducted across five localities in the northern Cordillera Central of Colombia, yielding 24 small mammal individuals (rodents, bats, and marsupials). Total genomic DNA was extracted from tissue samples and sequenced on Oxford Nanopore MinION devices using Flongle flow cells (R9.4.1), producing shotgun genomic sequencing data. Mitochondrial genomes were assembled from the raw ONT reads using a customized computational pipeline that included read quality filtering, mitochondrial read identification by alignment to reference mitogenomes, de novo assembly, and consensus generation. Species identification was performed by comparing assembled mitochondrial genomes against reference databases, with the entire analysis pipeline designed to run on standard laptop hardware for field-deployable applications.

3. Results

Figure 2. Nanopore-based species identification workflow for non-model Neotropical mammals. The pipeline combines MinION/Flongle shotgun sequencing, mitochondrial genome assembly from raw ONT reads, and a custom computational classification system for rapid, field-deployable species identification. Adapted from Velasquez-Restrepo et al. (2024), PeerJ, CC BY 4.0.

Key Findings

• Rapid species identification: The custom pipeline achieved accurate species-level identification for up to 75% of individuals in as little as 5 seconds of computational time per sample, demonstrating the feasibility of near-real-time field barcoding.
• Novel genomic resources: Three new mitochondrial genomes and eight new molecular sequences were generated for six species, expanding the reference database available for Neotropical mammal identification.
• Non-invasive potential: The shotgun sequencing approach required no species-specific PCR primers, enabling identification from any tissue type without prior knowledge of the sample's taxonomic identity.
• Field-deployable: The entire workflow — from MinION sequencing through bioinformatics analysis — was designed to operate on portable, low-cost hardware without internet connectivity, making it suitable for field stations and remote biodiversity monitoring sites.

4. Conclusions

This study demonstrates that Nanopore long-read sequencing, combined with a custom bioinformatics pipeline, provides a rapid, accurate, and field-deployable solution for species identification of non-model mammals. The approach is broadly applicable to other vertebrate and invertebrate taxa and is particularly valuable for biodiversity surveys in megadiverse tropical regions where rapid species assessment is urgently needed for conservation planning.

When to Choose Long Amplicon Sequencing — and When Alternative Methods May Be More Suitable

Choose long amplicon sequencing when:

• Full-length gene resolution is required for your markers — DNA barcoding (COI, rbcL, matK, ITS), transgene verification, and haplotype phasing benefit from single-molecule reads that span the complete target region without tiled assembly
• Your species lacks a reference genome or optimized primers — Long amplicon sequencing works on any species; we perform custom PCR optimization for non-model organisms, rare species, and environmental samples where off-the-shelf primers may not be available
• You need to multiplex many samples or multiple target regions — PacBio 384-sample barcoding and ONT 96-sample native barcoding enable cost-effective pooled sequencing of large sample sets or multi-locus panels in a single run
• Haplotype-resolved analysis of multi-kb loci is needed — Single-molecule reads spanning 1–15+ kb preserve phased haplotype information, enabling direct observation of heterozygous variants without statistical inference
• Your project requires both discovery and validation in a single workflow — High-accuracy PacBio HiFi consensus sequences serve as both discovery and validation data, eliminating the need for separate Sanger confirmation of key findings

Consider alternative methods when:

• Only short gene fragments (<500 bp) need to be screened — For small amplicons, short-read sequencing (Illumina MiSeq) may be more cost-effective, particularly for projects with very large sample sizes or established short-read workflows
• Genome-wide coverage, not targeted analysis, is required — Whole-genome sequencing (short or long read) or reduced-representation methods (GBS, RAD-seq) may be more appropriate for genome-wide marker discovery or population genomics
• RNA-level analysis of isoform diversity is needed — If the question involves alternative splicing, isoform discovery, or transcript-level quantification, our Nanopore Full-Length cDNA Sequencing or PacBio RNA Sequencing services are more appropriate
• Sample DNA is severely degraded — Highly fragmented DNA (average fragment size <1 kb) may not support amplification of long targets (>3 kb); in such cases, short-amplicon approaches or target capture methods may be necessary

CD Genomics provides free project consultation to help determine whether long amplicon sequencing is the right approach for your animal or plant research project. Contact our scientists to discuss your species and target regions.

Sample Deliverables for Animal and Plant Long Amplicon Sequencing Projects

1. High-accuracy consensus sequences (FASTA format) for each amplicon target, with per-base quality scores and coverage statistics — suitable for GenBank submission and phylogenetic analysis.

2. Multiple sequence alignment and maximum-likelihood phylogenetic tree (Newick format) with bootstrap support values, including species-level taxonomic labels and outgroup rooting.

3. Comprehensive variant report (VCF format) with SNP, InDel, and STR calls for population genetics analysis, including allele frequency tables and haplotype assignments.

4. Full project report in PDF format documenting all methods, QC metrics, analysis parameters, and results — designed for inclusion in manuscripts and grant reports.

Figure 3. Representative deliverable formats for animal/plant long amplicon sequencing projects. Left: consensus sequence alignment and phylogenetic tree for species identification. Center: variant detection and genotyping results for population-level analysis. Right: species-level taxonomic classification report with confidence scores. AI-generated representative data.