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Sequencing and bioinformatics from microbial genomics experts
Our short read sequencing service provides 150bp paired end reads.
Short reads can be useful for a range of applications, including:
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 Box 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
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:
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 Box in FASTQ format, along with methods and QC reports.
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.
Please contact us for pricing of long read (Nanopore) strain typing.
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.
16S amplicon sequencing is useful for examining the species diversity and abundance of a microbial community. Some examples of complex microbial communities:
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:
To generate high-quality reference assemblies, we recommend pairing this sequencing bundle with bioinformatics services:
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 Box in FASTQ format, along with methods and QC reports.
Our RNA sequencing services provide 150bp paired end reads.
Because eukaryotic mRNA has a poly-A tail, we use the poly-A enrichment method to reduce the quantity of rRNA in the sample and enrich for mRNA. Please note that poly-A enrichment is not suitable for bacterial samples.
RNAseq is useful for:
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 >50ng/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 Box in FASTQ format, along with methods and QC reports.
Our RNA sequencing services provide 150bp paired end reads.
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:
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 >50ng/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 Box in FASTQ format, along with methods and QC reports.
Have a prepared library pool that is compatible with Illumina? We can run a dedicated flow cell for you! Choose from a wide variety of flow cell configurations that are available for the NextSeq2000.
Pricing includes QC of your sample (Qubit and TapeStation), dilution to loading concentration, demultiplexing analysis using the customer-provided sample sheet, and data delivery. Library pooling is available as an add-on service and will be billed hourly.
You must provide the following information:
Need even more long reads? Purchase a full GridION flow cell!
Expected yield is 10-20+Gb, depending on library quality and fragment length. You can estimate the number of reads by dividing the total expected yield by your fragment length. We use the ONT super-accurate basecalling model, v14 reagents, and an R10 flow cell to give you the most accurate data possible.
Pricing includes QC (Qubit and TapeStation), PCR-free library prep for one sample (either native ligation or rapid transposase), and data delivery. Contact us for specialized library preps and run conditions.
We now offer adaptive sampling methods to enrich for coverage in regions of interest! This method is especially useful for applications like host enrichment/depletion and deeper sequencing of target regions comprising 0.5-5% of whole genomes. This service requires extra handling steps that are billed hourly.
We currently offer DNA extraction for pure microbial cultures (BSL2 or below), which can be sent to us as either frozen cell pellets or sealed, pre-grown agar plates.
We use bead beating lysis to crack even the toughest microbes!
If sending cell pellets, please aim for 50-100mg. If your species is known to present challenges for extraction, please contact us before ordering this service so that we can confirm availability of the necessary reagents and equipment.
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 >90%, and elution can be done in as little as 20ul.
SPRI bead cleanup can help remove common contaminants, such as:
We are proud to offer world-class bioinformatics support! We can work with you from initial data analysis through producing publication-ready figures. Our experts 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.
Custom bioinformatics
Match our microbial genomics expertise to your exact needs. For a custom quote contact us or select custom bioinformatics at checkout.
Variant calling
Our variant calling service includes mapping of short reads against a reference genome. This reference genome could be a published sequence in a public database or could be generated from a sequenced sample (see Genome assembly service below). Our service uses the best-of-class Breseq software to provide you with intuitive insights into the genetic differences between your sample and the genome reference. This analysis is particularly useful for sets of closely related genomes, like those generated during short-term experimental evolution.
Included with the standard service ($20/sample):
Included with deluxe service ($40/sample):
Genome assembly
Our genome assembly service includes reference-based (if reference sequence is available) or de novo assembly of sequenced short and/or long reads.
Amplicon/microbiome profiling
Our amplicon/microbiome profiling services includes analysis of amplicon reads using Qiime2 and DADA2. This analysis includes decontamination, generation of OTU/ASV tables, taxonomic profiling, and basic diversity estimates (including community ordination and dissimilarity analyses) using the R package Vegan.
Differential expression analysis
We offer basic analyses of RNA sequencing data. Specifically, we 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.
Annotation
Prediction of genes and gene functions using the BAKTA software. Gene calling is carried out using Prodigal. A variety of databases and software are then used for identification of gene functions, protein domains, and metabolic pathways. Genes are compared against Pfam and TIGRFAMS databases, as well as KEGG Orthology, COGs, CAZy, and FeGenie. Enzyme Commission numbers are also assigned. Possible contaminants are identified using the SprayNPray software. A variety of data file types are included in the provided output, including human-readable spreadsheets and downstream analysis-friendly common file types (e.g., GFF, GenBank, FASTA formats, and image files).
Phylogenomics and tree-building
We also offer phylogenomic analysis of genomes sequenced and assembled through SeqCoast. This includes identification of close relatives and more distantly related representative genomes. We use GToTree to generate a multi-sequence alignment of concatenated proteins, and then use RAxML to produce a bootstrapped maximum-likelihood phylogenomic tree.
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.
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.
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