Custom

Glad you asked! When you are ready to proceed, please submit the Custom Project Intake Form. If you need extra help planning out your sequencing assay before getting started, please email support@plasmidsaurus.com for assistance.

The cost for each Custom Sequencing project starts at $500 for the 1st Gb (gigabase) of total raw data, then adds $50 for each additional 1 Gb. If barcodes are required for multiplexing, this adds a $50 charge per sample:

Project Cost = $500 base price for 1st Gb data + $50 for each extra Gb data + $50 x number of samples

We calculate your project price when you submit the Custom Project Intake Form. Note that each batch of shipped samples starts again at the base price of $500, so if you are prioritizing cost over speed, please consider reducing the number of batches you ship us to minimize your overall project price. 

In the vast majority of cases, we deliver Custom Sequencing results within 3-5 business days of receipt of your samples at the Eugene, Oregon lab. Projects that require very large amounts of data, or add on extra services such as DNA extraction or bioinformatics, may take longer.

In most cases, we deliver only the raw sequencing reads (in .fastq format) for Custom projects. Any analyses (demultiplexing internal barcodes, generating consensus sequences, binning or aligning variants, etc.) must be done by the researcher, unless we specifically agree to perform analysis during project setup.

Due to variation in sample quality and other stochastic factors, the samples within a project may produce slightly different amounts of data. Any sample in the project that produces at least 75% of its data target will be denoted “complete” in your results and is not eligible for free rerun.

Any sample that produces less than 75% of its data target will be denoted “fail” in your results and will be automatically resequenced at no additional charge in an attempt to collect more data. If the sequencing performance on the initial run is particularly poor, we may also ask you to ship us a replacement sample for the rerun.

Note: If your project requires that all samples achieve a minimum data target (i.e. you cannot accept any samples that produce only 75% of the average data target), please let us know during project set-up and we will increase the data target such that obtaining 75% of the average target would still meet your minimum requirement. You will be charged according to this increased target.

We construct an Oxford Nanopore amplification-free long-read sequencing library using the newest v14 library prep chemistry.

• For circular dsDNA, we use tagmentation to linearize each molecule in a sequence-independent manner.
• For linear dsDNA, we use sequence-independent end-ligation.
• For genomic dsDNA, we use sequence-independent tagmentation that minimally fragments the gDNA (unless you specifically request that we switch to end-ligation instead).

We then sequence the library with a primer-free protocol using the most accurate R10.4.1 flow cells to collect the amount of data that you specifically request. Please do not ship any primers with or mixed into your samples.

You will receive only the raw sequencing reads (in .fastq format) for Custom Sequencing projects, unless we specifically agree to perform an analysis during project set-up.

We require a minimum raw read Qscore of 10 (90% accuracy) during sequencing, although most raw reads are above Q20 (99% accuracy). We use the super-accurate basecalling model.

Since we typically do not perform any further analysis of the raw reads for Custom Sequencing, the final accuracy of your own analysis will depend on your analysis pipeline and quality filtering.

For variant libraries
Total Data Required = Number of samples x Insert length x Number of variants (barcodes, mutants, etc.) x Coverage required per variant

For genomic sequencing
Total Data Required = Number of samples x Expected genome size x Coverage required per genome

We fluorometrically quantify your input DNA and multiplex the samples in equimolar ratio for sequencing. Due to variation in sample quality and other stochastic factors, the samples may still produce slightly different amounts of data. Every custom project does include a free increase to the project data target to account for some expected variability, but lower quality samples may still fail to achieve the target. Therefore, the data target should be interpreted as an average per-sample target of basecalled data, where some samples may end up higher and others may end up lower.

If you need different amounts of data for each sample, you can request this! Just let us know when you set up your order. 

In most cases, we deliver only the raw sequencing reads (in .fastq format) for Custom projects. Any analyses (demultiplexing internal barcodes, generating consensus sequences, binning or aligning variants, etc.) must be done by the researcher, unless we specifically agree to perform analysis during project setup.

We do not accept RNA for Custom Sequencing at this time, so you would need to convert your RNA into double-stranded cDNA before shipping it to us.

Single-stranded DNA is not currently a supported application for Custom Sequencing, but we do have some beta methods in development that we might be able to test out. Please email support@plasmidsaurus.com to inquire about ssDNA sequencing options.

Yes! We perform do a wide range of special workflows, such as delivering POD5 files or methylation BAM files, Adaptive Sampling to enrich or deplete a sequence of interest, read filtering to retain only specific lengths, or swapping out ONT long-read data for Illumina short-read data. If you're interested in any of these or any other special workflows, please explain the nature of your special request when you submit the Custom Project Intake Form. Note that some types of requests may incur an additional fee.

Step 1: Register for a Plasmidsaurus account 
 

Step 2: Find your local dropbox for fast free shipping 

Or if preferred, you can ship samples directly to the Eugene, Oregon lab at room temp: 

Plasmidsaurus
1850 Millrace Drive, Suite 200
Eugene, OR 97403
 

Step 3: Submit the Custom Project Intake Form 

Use this form to request the Custom QR code that needs to be included with your shipment.

For Fiber-seq QC for 6mA service ($75/sample for approx. 100 Mb data):

• Enter “Fiber-seq QC for 6mA” for Project Description
• Enter “% 6mA analysis by Plasmidsaurus” for Data Analysis
• Select “Genomic dsDNA” for Topology
• Enter the average DNA fragment length as determined by your gel electrophoresis for Average Length -- do NOT enter size of genome
• Enter “200 bp” for Minimum Length
• Select “(A) I will provide the amount of raw data required (in Megabases, Mb or Gigabases, Gb) per sample” for data requirement, then select “Mb” and enter “100” (for shallow sequencing*)
• Enter “6mA all contexts basecalling” in Special Requests 

For Fiber-seq whole genome service: ($1750/flow cell for approx. 50-100 Gb data):

• Enter “Fiber-seq whole genome” for Project Description
• Enter “Raw FASTQ & BAM reads only” for Data Analysis
• Select “Genomic dsDNA” for Topology
• Enter the average DNA fragment length as determined by your gel electrophoresis for Average Length -- do NOT enter size of genome
• Enter “200 bp” for Minimum Length
• Select “(A) I will provide the amount of raw data required (in Megabases, Mb or Gigabases, Gb) per sample” for data requirement, then select “Gb” and enter either “100” (for 30x coverage*, 1 sample per flow cell) or “50” (for 15x coverage*, 2 samples per flow cell)
• Enter “6mA, 5mC, & 5hmC all contexts basecalling” in Special Request

* Actual data yields for genomic sequencing are dependent on DNA quality/purity and are not guaranteed

NOTE: It may take up to 2 business days to receive a reply to your Custom Project Intake Form.
 

Step 4: Quantify, label, package, and ship your purified genomic DNA samples 

For Fiber-seq QC for 6mA service: 
Input of purified genomic DNA required per sample = minimum 20 µL @ minimum 15 ng/µL 

For Fiber-seq whole genome service: 
Input of purified genomic DNA required per sample = minimum 20 µL @ minimum 50 ng/µL 

NOTE: If you may end up wanting both the QC for 6mA and the whole genome services on the same tube of DNA, please send the higher DNA amount that is required for whole genome. 

NOTE: DNA fragments that are shorter than 3 kb will NOT be sequenced with this method, so please do not send extremely degraded gDNA or intentionally sheared gDNA.

Yes, this can be requested as a new Custom category called “MAG Sequencing”! We ligate ONT adapters onto the ends of the extracted gDNA and then collect ONT long reads, available at two data tiers:

• 30 Gbases for lower complexity communities (for assembling genomes at an abundance >1%) = $600/sample 
• 100 Gbases for higher complexity communities (for assembling genomes at an abundance >0.3%) = $1200/sample

The DNA input can either be your own extracted gDNA samples (minimum 60 µL @ minimum 50 ng/µL), OR you can request on-site extraction from Zymo Shield-preserved raw biological material, available with two methods:

• $15/sample = designed to maximize overall DNA yield from low biomass material via vigorous bead bashing and chemical lysis. Available for any sample type indicated in our Microbiome Extraction Sample Prep instructions, ideal for low biomass samples like soil.
• $100/sample = designed to maximize the DNA fragment length from high biomass material via the "Three Peaks" progressive extraction strategy. An initial gentle lysis to isolate high-molecular-weight DNA from easily-lysed species (providing longer reads for complex repeat regions and circularized contigs) is followed by more aggressive mechanical lysis to capture gram-positive and other difficult-to-lyse species (ensuring that assembly is taxonomically representative and that no functional niche is overlooked). Currently available for feces only, prepared per our Microbiome Extraction Sample Prep instructions. 

For data analysis, we run myloasm to de novo assemble MAGs in the sample. We return a fasta file of the assembled genomes and contigs and a report that lists the length and read depth of the assembled sequences. You will also receive all of the raw reads (FASTQ) from the sequencing run. The turnaround time is ~5 business days for the 30 Gb tier and ~10 business days for the 100 Gb tier.

Yes, a new analysis method that we call “RARE” (Rejecting Abundant Reads for Enrichment) is now available as an add-on for MAG Sequencing of fecal samples!

High-abundance species can consume significant sequencing bandwidth, leaving rare but significant taxa under-sampled. This method leverages ONT’s adaptive sampling workflow to focus the reads on less-abundant species and generate more long contigs from complex microbiomes that are often hampered by the extreme dynamic range of species abundance. 

Our RARE method maximizes the utility of every nanopore on the flow cell. First, preliminary sequencing generates high-depth contigs of the most abundant species via myloasm assembly. These are used as a "reject list" for ONT’s adaptive sampling algorithm, such that if a new read matches one of the initial contigs, the pore ejects the molecule and immediately begins sequencing a new fragment. This prevents the over-sequencing of abundant species and redirects capacity toward the low-abundance taxa.

To demonstrate this, we performed “Three Peaks” extraction and MAG Sequencing of the ZymoBIOMICS Fecal Reference, both with and without the RARE method. We found that using RARE generated >30% more contigs and high-quality contigs, indicating that RARE supported by progressive extraction can capture a more complete and contiguous picture of complex microbial communities.

Important note! Because the RARE method requires very high DNA quantity & quality from a community with sufficient microbial diversity, it is only available for fecal samples that are extracted on site by Plasmidsaurus with the progressive “Three Peaks” method. The additional costs for fecal extraction and RARE analysis are included in this single available tier, with a turnaround time of ~10 business days:

• 100 Gbases * = $1500/sample
  * Some samples with high levels of rejected reads will not reach this target, but will be even more enriched for low-abundance species.

STEP 1: Submit the Custom Project Intake Form.
Use this form to request the Custom QR code that needs to be included with your shipment.

For MAG Sequencing only:

• Enter “MAG Sequencing of [sample type = soil, feces, etc]” for Project Description
• Enter “myloasm assembly” for Data Analysis
• Select “Genomic dsDNA” for Topology
• Enter the average DNA fragment length as determined by your gel electrophoresis for Average Length (or enter TBD if you are requesting on-site DNA extraction)
• Enter “200 bp” for Minimum Length
• Select “(A) I will provide the amount of raw data required (in Megabases, Mb or Gigabases, Gb) per sample” for data requirement, then select “Gb” and enter either “30” or “100” *
• If you’d like to include on-site DNA extraction for your raw biological material, enter one of the following in Special Requests:
  • Add vigorous low-biomass extraction at $15/sample 
  • Add “Three Peaks” high-biomass extraction at $100/sample 

For MAG Sequencing + RARE method (only available for on-site fecal extractions):

• Enter “MAG Sequencing of fecal samples + RARE method + three-peaks extraction” for Project Description
• Enter “myloasm assembly + RARE” for Data Analysis
• Select “Genomic dsDNA” for Topology
• Enter “TBD” for the average DNA fragment length
• Enter “200 bp” for Minimum Length
• Select “(A) I will provide the amount of raw data required (in Megabases, Mb or Gigabases, Gb) per sample” for data requirement, then select “Gb” and enter “100” *

NOTE: It may take up to 2 business days to receive a reply to your Custom Project Intake Form.

* Actual data yields for genomic sequencing are dependent on DNA quality/purity and are not guaranteed

STEP 2: Prepare and ship your samples with the provided Custom QR code.

For MAG Sequencing only:

Input required per sample for purified genomic DNA = minimum 60 µL @ minimum 50 ng/µL. Quantify, label, package, and ship gDNA per our Custom Sample Prep instructions. If you are not able to obtain this required amount of DNA, please send at the minimum concentration listed but in a lower total volume.

OR:

Input required per sample for raw biological material = any Zymo Shield-preserved sample type indicated in our Microbiome Extraction Sample Prep instructions, placed in 2 mL screw cap tubes

For MAG Sequencing + RARE method (fecal material only):

Input required per sample = 100-250 mg feces in 500 µL Zymo Shield, prepared per our Microbiome Extraction Sample Prep instructions and placed in 2 mL screw cap tubes