Sequence Smarter. Discover More.

Our short read sequencing service provides 150bp paired end reads.

Short reads can be useful for a range of applications, including:

• genotyping clones
• whole population sequencing
• phage sequencing
• metagenomic sequencing
• amplicon/microbiome profiling

Advantages of short read sequencing include high read accuracy (Q30+) and high throughput, both of which are important for applications like variant calling.

For an extra fee we can use a PCR-free library prep method to support your sensitive applications. This method requires higher DNA input (>20ng/ul), but it avoids PCR bias and reduces overall sequence error.

Data will be delivered via SFTP, Box, or AWS S3, (your choice)  in FASTQ format, along with methods and QC reports.

If providing gDNA, we request a concentration of >10ng/ul in a volume of at least 30ul. We prefer that sample concentrations be quantified by Qubit or other fluorometry-based techniques, as Nanodrop methods tend to overestimate quantity. Use water or dilute TE for elution.

For phage sequencing, we recommend our Small, 200 Mbp Illumina short read service.

SeqCoast is an Oxford Nanopore Service Certified laboratory! Our certification ensures that SeqCoast can offer the highest quality services using Oxford Nanopore Technologies.

Our long read sequencing service provides reads up to 100kbp (occasionally much longer) in length. To maximize read length we strongly recommend using our extraction service, because long DNA fragments may shear during shipping and with some extraction methods.

Long reads can be useful for a range of applications, including:

• sequencing longer amplicons, such as full length 16S
• resolving repetitive plasmids or viruses
• spanning difficult to map/assemble regions
• establishing linked mutations even in a mixed population
• resolving complex metagenomes
• identifying base modifications, like methylation

Advantages of long read sequencing include library prep method flexibility (can sequence native strands!), the ability to sequence low-diversity amplicons, and drastic improvements to genome assemblies, especially when combined with short reads.

Data will be delivered via SFTP, Box, or AWS S3 (your choice), in FASTQ and POD5 format, along with methods.

If providing gDNA, please send >40ng/ul in a volume of at least 60ul. We prefer that sample concentrations be quantified by Qubit or other fluorometry-based techniques, as Nanodrop methods tend to overestimate quantity. Use water or dilute TE for elution.

Our convenient and comprehensive strain typing service bundles bead-beating extraction, short read sequencing, and bioinformatics analysis to produce reports on the MLST type of your strain of interest.

This service is especially useful for projects involving epidemiological surveillance, because it can help reveal whether strains of the same species are closely related (suggesting an outbreak) or distantly related (suggesting independent origins).

“Strain typing methodologies have recently undergone a paradigm shift as whole-genome sequencing (WGS) has become cheaper and more accessible to clinical and public health laboratories. WGS provides unmatched resolution and discriminatory power for highly related strains, and it has significant potential for outbreak detection, epidemiological surveillance, and infection control strategies.” Simar, et al, 2021

Please submit frozen cell pellets (50-100mg) or sealed, pre-grown agar plates.

Amplicon sequencing is a good first step to understanding which microbes are present and their relative abundance. To gather even more information about mixed-species populations, consider using shotgun sequencing and combining with our comprehensive metagenome analyses.

• Our 16S Illumina service provides 300bp paired end reads. Standard primers are 341F and 806R, which amplify the V3/V4 hypervariable regions.
• Our full-length 16S Nanopore service provides the entire 16S sequence, roughly 1500bp in length. Full-length 16S provides better species and strain-level resolution than Illumina-based datasets. Custom primers for ITS and other amplicons are available. Bulk pricing for full-length 16S starts at 12 samples, contact us for more information!

16S amplicon sequencing is useful for examining the species diversity and abundance of a microbial community. Some examples of complex microbial communities:

• Gut microbiome
• Soil communities
• Marine microbial communities
• Synthetic microbial consortia

Need analysis? Inquire about our amplicon bioinformatics reports that provide community composition and diversity metrics, in both table and graph formats.

If providing gDNA, we request a concentration of >10ng/ul in a volume of at least 30ul. We prefer that sample concentrations be quantified by Qubit or other fluorometry-based techniques, as Nanodrop methods tend to overestimate quantity. Use water as the diluent. Samples must be free of PCR inhibitors, including EDTA. Sequencing data will be delivered in FASTQ format, along with methods and QC reports.

This hybrid sequencing strategy is especially useful for building robust reference genomes; the long reads drastically reduce the number of contigs and the depth afforded by the short reads increases the confidence of each nucleotide position.

This bundled service provides:

• High molecular weight gDNA extraction
• >50X coverage for short reads (Illumina, 2x150bp)
• >30X coverage for long reads (Oxford Nanopore)

To generate high-quality reference assemblies, we recommend pairing this sequencing bundle with bioinformatics services:

• Assembly
• Annotation

To maximize length of long reads, we strongly recommend using our extraction service, because long DNA fragments may shear during shipping and with some extraction methods.

If providing gDNA, please send >40ng/ul in a volume of at least 60ul. We prefer that sample concentrations be quantified by Qubit or other fluorometry-based techniques, as Nanodrop methods tend to overestimate quantity. Use water or dilute TE for elution.

Data will be delivered via SFTP, Box, or AWS S3 (your choice) in FASTQ format, along with methods and QC reports.

Our RNA sequencing services provide 150bp paired end reads, and our RNAseq protocols are stranded.

Because bacterial mRNA does not have a poly-A tail, we use a method called ribodepletion to reduce the quantity of rRNA in the sample. Ribodepletion is also compatible with eukaryotic samples, but poly-A tail enrichment may be a better choice for these samples.

RNAseq is useful for:

• sensitive, accurate gene expression analyses that connect the genome to active transcription
• metatranscriptomics to get a snapshot of a population’s gene expression

RNAseq agnostically captures the full transcriptome, unlike probe-based methods such as microarrays. While it may be helpful for downstream analyses, no reference sequence is necessary to perform RNAseq.

We recommend pairing RNAseq with differential expression analysis.

Please send >25ng/ul of RNA in a volume of at least 20ul, shipped frozen on dry ice. We prefer that sample concentrations be quantified by Qubit or other fluorometry-based techniques, as Nanodrop methods tend to overestimate quantity. DNAse treatment during extraction is highly recommended. For optimal results, the RIN should be at least 7.

Data will be delivered via SFTP, Box, or AWS S3 (your choice) in FASTQ format, along with methods and QC reports.

Our extraction service leverages bead-beating lysis to ensure efficient processing of even the toughest microbes! Our standard DNA extraction service accommodates:

• Organisms that are BSL2 and below
• Frozen cell pellets with a wet weight of ~50-100mg, OR
• Sealed, pre-grown agar plates with robust growth

Note that if you are shipping sporulating fungi for extraction we request that you send a frozen cell pellet, rather than pre-grown agar plates.

Need DNA extracted from more complex samples?

For challenging extractions, we offer higher-level services tailored to your unique needs. Please contact us for custom extraction needs!

We now offer an SPRI bead clean and concentrate service! This is a great solution when your samples are either too dilute or are suspected to contain contaminants from the extraction process that will interfere with sequencing. Sample recovery by SPRI beads is usually >80%, and elution can be done in as little as 20ul.

SPRI bead cleanup can help remove common contaminants, such as:

• excessive EDTA
• salts
• phenol
• ethanol/isopropanol

We are proud to offer a focused slate of value-packed bioinformatics reports at highly competitive prices. We still provide custom bioinformatics services for customers with high-touch needs.

Our core bioinformatics analyses are developed and processed on the secure AWS cloud-based platform using Seqera’s NextFlow Tower, and our version control is maintained using GitHub.

Prokaryote Assembly and Annotation

List Price:   $28.00 but we offer custom pricing on bulk orders – please contact us

SeqCoast offers a comprehensive Prokaryote Genome Assembly and Annotation service. Our pipeline is designed to handle both short and long-read sequencing data, making it ideal for researchers studying bacterial genomes using Illumina, Oxford Nanopore, or hybrid approaches. Our robust workflow ensures accurate assembly and annotation, providing high-quality results for both small- and large-scale projects.

What We Deliver:

1. Quality Control and Data Processing:
   • Short Reads: FastP and FastQC ensure the quality of your Illumina data.
   • Long Reads: PoreChop and NanoPlot assess and improve your Nanopore data.
   • Contamination screening is performed using Kraken2 to provide taxonomic insights at the read level
2. Genome Assembly:
   • Short-Read Assemblies: Generated using SPAdes wrapped in Unicycler.
   • Long-Read Assemblies: Produced with Miniasm wrapped in Unicycler.
   • Hybrid Assemblies: Combines short and long reads with SPAdes and Miniasm for the most complete assembly.
3. Assembly Polishing:
   • Short Reads: Polished using Pilon.
   • Long and Hybrid Reads: Polished with Racon and additional polishing with Medaka for long reads.
4. Assembly Quality Assessment:
   • The quality of your genome assembly is evaluated using QUAST, providing reports with detailed metrics on assembly quality.
5. Genome Annotation:
   • Annotated using Bakta, we provide detailed reports, including:
     1. GFF3 files: Annotated gene features
     2. FASTA files: Protein sequences and nucleotide sequences
     3. TXT files: Summary reports of the annotations

6. Comprehensive Reports:
   • A MultiQC report compiles all quality control metrics across samples into a single interactive HTML document for easy review.

Bacterial Variant Calling

List Price:   $15.00 but we offer custom pricing on bulk orders – please contact us

SeqCoast offers high-quality bacterial variant calling services, designed to help researchers identify mutations, structural variations, and copy number variations (CNVs) in clonal bacterial genomes or genetically diverse samples. Our workflow accommodates both Illumina and Oxford Nanopore sequencing data, offering flexibility and precision in variant detection.

Bacterial variant calling typically requires a minimum of 30x mean coverage, and higher coverage if you are surveying mixed populations. Converting this to our offerings, multiply [desired coverage] x [genome size, in Mb].

Note: This pipeline is only appropriate for genomes with a closely related reference genome. Don’t have one? Use our Hybrid Sequencing Service in combination with Assembly and Annotation to generate a high-quality de novo reference genome for your wildtype strain.

What We Deliver:

1. Quality Control and Read Filtering:
   • Short Reads: Processed with Trimmomatic for trimming and filtering to ensure high-quality data.
   • Long Reads: Processed with PoreChop for optimal long-read performance.
2. Read Mapping:
   • Reads are mapped to your provided reference genome using Bowtie2, an industry-standard tool for accurate read
3. Variant Detection:
   • Variants are called using Breseq, a robust pipeline that identifies mutations such as SNPs, insertions, and deletions.
   • For diverse (non-clonal) populations, mutation frequency estimates provide a deeper understanding of genetic diversity.
4. Copy Number Variation (CNV) Analysis:
   • CNV prediction is performed across samples, helping you detect and quantify structural variations in your data.
5. Comprehensive Output Files:
   • Mutation Summaries: Mutation profiles across samples are provided in both tabular (CSV) and visual formats (HTML).
   • CNV Results: Copy number variation is detailed in CSV reports for each sample.
   • Logs and Raw Outputs: Access to all Breseq output files and logs for further analysis or verification.
6. User-Friendly Report:A viewable HTML summary of mutation results, with links to evidence supporting each mutation, is included for easy interpretation.

Short Read Amplicon Analysis

List Price:   $25.00 but we offer custom pricing on bulk orders – please contact us

Our short-read Amplicon Sequencing Analysis pipeline is robust, scalable, and optimized for both small and large datasets. With automated quality control, accurate ASV inference, and comprehensive taxonomic analysis, our amplicon sequencing service is designed to deliver results you can trust. Whether you are exploring microbial diversity, identifying species, or conducting ecological studies, our service delivers high-quality, reproducible results that meet the needs of both new and experienced researchers.

What We Deliver:

1. Quality Control and Data Processing:
   • All sequences undergo thorough quality checks and primer trimming to ensure data integrity.
2. Amplicon Sequence Variant (ASV) Inference:
   • Using DADA2, we accurately infer amplicon sequence variants (ASVs), eliminating errors while preserving biological diversity in your samples.
3. Taxonomic Classification:
   • Taxonomic classification is performed using established databases such as Silva for 16S rRNA, Pr2 for 18S rRNA, and Unite for ITS.
   • Contact us for a quote to curate custom reference databases for specialized applications.
4. Diversity and Phylogenetic Analysis:
   • We provide both alpha and beta diversity indices to assess microbial diversity within and between samples based on metadata that you provide. Phylogenetic trees are used to help visualize evolutionary relationships between ASVs.
5. Interactive Reports and Data Files:
   • You will receive a comprehensive analysis package, including:
     1. Interactive abundance bar plots
     2. Rarefaction curves
     3. Diversity metrics (Shannon, Faith’s PD, Bray-Curtis, etc.)
     4. Data files are provided in various formats (TSV, FASTA, BIOM) for further downstream analysis.

6. Phyloseq R Objects:
   • We provide Phyloseq R objects for custom downstream analysis and visualization in R.

Long Read Amplicon Analysis

List Price:   $25.00 but we offer custom pricing on bulk orders – please contact us

SeqCoast provides high-quality long-read 16S amplicon sequencing analysis, tailored for Oxford Nanopore sequencing data. Our pipeline generates detailed taxonomic profiles, offering robust insights into microbial community composition with the added advantage of longer amplicon lengths. Because long-read amplicons are able to span the entire 16S rRNA sequence, this analysis offers higher taxonomic resolution than short-read amplicon approaches, frequently achieving resolution at the species (and sometimes strain!) level.

What We Deliver:

1. Taxonomic Classification:
   • Reads are classified taxonomically using Minimap2 against the NCBI Targeted Loci database to generate precise taxonomic profiles.
   • Species-level taxonomic assignments provide a high-resolution view of microbial diversity.
2. Comprehensive Report:
   • You will receive a detailed HTML report summarizing the results, including taxonomic profiles for each sample.
   • A TSV file is provided that contains per-species abundance counts and can be used for downstream analysis.

Bacterial Profiling and Characterization

List Price:   $40.00 but we offer custom pricing on bulk orders – please contact us

Useful for: Strain Typing, Surveillance, and Outbreak Detection

SeqCoast offers an advanced bacterial profiling and characterization service, designed to provide comprehensive insights into bacterial genomes and longitudinal monitoring of bacterial populations.

Key Applications:

• Strain Typing: Accurately identify and differentiate bacterial strains for targeted research and interventions.
• Antimicrobial Resistance Studies: Predict resistance phenotypes and discover new resistance mechanisms.
• Virulence Factor Analysis: Identify and characterize virulence markers for pathogenicity studies.
• Contamination Monitoring and Source Tracing: Pinpoint the source and characterize the spread of bacterial contaminants in your facility or in the environment.

Advanced Longitudinal Applications:

• Surveillance and Monitoring: Track bacterial populations over time to detect emerging threats and evolutionary shifts.
• Outbreak Detection: Rapidly identify, track, and characterize potential outbreak clusters.

Our innovative approach goes beyond a single time-point – we retain your data and perform iterative analyses, allowing for continuous monitoring of bacterial populations over time. Contact us to discuss how our longitudinal surveillance service can strengthen your monitoring and research initiatives.

Our Comprehensive Service Includes:

• Advanced quality control and read filtering
• Precise taxonomic identification and abundance estimation
• High-quality genome assembly and annotation
• Multi-locus sequence typing (MLST)
• Detection of antimicrobial resistance genes and virulence factors
• Plasmid and mobile genetic element identification
• Susceptibility and resistance phenotype prediction for select species
• Iterative analysis for continuous monitoring and outbreak detection

What We Deliver:

1. Quality Control and Read Filtering:
   • Quality control, filtering, and trimming of raw reads are conducted using industry-standard tools (Fastp, Trimmomatic, Rasusa).
   • The pipeline provides both pre- and post-filtering QC reports to ensure data integrity.
2. Taxonomic Identification and Abundance Estimation:
   • Reads are classified taxonomically using Kraken2 and Bracken, providing detailed taxonomic profiles with abundance estimation.
   • Reports are delivered in multiple formats, including BIOM for seamless integration with downstream analysis tools.
3. Genome Assembly and Quality Control:
   • We assemble genomes using SPAdes, Unicycler, or Shovill, followed by comprehensive quality control with QUAST, BUSCO, and CheckM.
   • Assembly coverage is mapped using BWA-MEM2 and further analyzed for structural integrity.
4. Annotation and Typing:
   • Annotated genomes include functional features identified by Prokka and Barrnap, with multi-locus sequence typing (MLST) performed using PubMLST schemes.
   • Plasmid content and other mobile genetic elements (MGEs) are identified using MOB-suite.
5. AMR and Virulence Marker Detection:
   • AMR genes are detected with AMRFinderPlus and ResFinder, while virulence markers are screened using the Virulence Factor Database (VFDB).
   • Plasmid-mediated resistance is flagged for comprehensive reporting.
6. Susceptibility and Resistance Phenotype Prediction:
   • For select species, susceptibility and resistance phenotypes are predicted using ResFinder and Mykrobe, providing insights into antimicrobial resistance.
7. Outbreak Detection and Iterative Analysis:
   • Our pipeline supports iterative runs, enabling continuous monitoring of bacterial populations and rapid detection of outbreak clusters.
   • Samples are clustered based on SNP distances, with detailed clustering reports provided for each iteration.
8. Comprehensive Reports and Data Summaries:
   • Per-sample reports in Excel and JSON formats summarize all key metrics, including SNP distances and outbreak tracking.
   • Combined summaries across samples offer detailed insights for outbreak detection and longitudinal studies.

Add-ons:

• ANI Analysis:

Working with unknown isolates?  We use Sourmash to identify the most closely related RefSeq genomes to your query sequence based on Average Nucleotide Identity (ANI). This analysis includes a comprehensive CSV report for easy interpretation and downstream integration.

Custom Bioinformatics

For pricing your custom services, please contact us.

Examples include differential expression, metagenomics, transcriptome assembly, pangenomic analysis, etc.

Our bioinformaticians can work with you from initial data analysis through producing publication-ready figures. They will tailor the level of support to your needs – whether you are a beginner that needs help with the very basics or an experienced power-user looking to reduce your workload.

Match our microbial genomics expertise to your exact needs.

• Differential expression analysis

We offer basic analyses of RNA sequencing data. Specifically, we use Kallisto to estimate gene expression levels.  We also use DESeq2 to estimate differential expression between samples and process the output into a user-friendly document that includes volcano plots for user-specified comparisons, as well as data files that are amenable to additional downstream processing.

Our differential expression analysis services are typically charged on a per-sample basis. Please reach out to us describing your experimental design if you are unsure what quantity to order for differential expression analysis or if you are interested in comparing more than two variables.

• Phylogenomics and tree-building

We offer a variety of analyses for metagenomic studies. These range from assembly of sequenced reads into longer contiguous sequences (called contigs or scaffolds) to reconstruction and curation of metagenome-derived genomes.

• Metagenome assembly and binning

We offer a variety of analyses for metagenomic studies. These range from assembly of sequenced reads into longer contiguous sequences (called contigs or scaffolds) to reconstruction and curation of metagenome-derived genomes.

• • Assembly: Megahit and metaSPAdes are used to assemble high-quality reads.
  • Assembly with binning and bin curation: Assembly with Megahit and metaSPAdes, followed by unsupervised binning with MetaBAT. Draft genome bins are then manually curated using a variety of software, including SprayNPray, BinaRena, and CheckM.
• Construct validation

As a service, we bioinformatically validate genetically engineered samples and genomes. We quickly confirm the presence or absence of knock-ins and knock-outs, and identify other (off target) mutations.

• Consultation

All bioinformatics analyses include a summary report that describes the provided files, as well as the steps taken to generate those files. To gain further insight and to plan new analyses, we also offer consultation (virtual/Zoom) sessions with a bioinformatics specialist.