Submit Revio samples

PacBio Revio bulk RNA IsoSeq sample submission

We are currently accepting samples for Bulk IsoSeq analysis using Revio.

Isoform sequencing requires a minimum of 300 ng RNA with a minimum concentration of 43 ng/uL. The RNA Integrity Number (RIN) should be > 7 for optimal results. Include a DNase I treatment in the RNA extraction protocol. Nanodrop reading should have A260/280 ratio of ~2.0 and an A260/230 ratio is ≥2.0. If using a phenol/chloroform based extraction method such as TRIzol, a bead cleanup is recommended to remove carryover contaminates that can interfere with the RT enzyme. If necessary, the gCore can do bead cleanup for a nominal fee. Submitting 500 ng is recommended when possible. Each run will give the equivalent of ~40 million full-length RNA strands (the exact number depends on cell type and RNA quality).

Pricing

The recommended batch size is 8 samples. This allows for sequencing at a depth of 5M reads per sample or 10M reads per sample. 5M reads per is recommended for isoform discovery and abundant transcripts. 10M reads per sample is recommended for quantifying differential expression of isoforms. More reads may be required for particularly rare isoforms or samples with high levels of RNA.

Current UCSD pricing is:

$488 per sample for 5M reads per sample in an 8-sample batch.
$688 per sample for 10M reads per sample in an 8-sample batch.

The price includes library preparation and sequencing

For other read depths and sample numbers, use gCore bulk IsoSeq bulk RNA Price Calculator.
How pricing is calculated.

Email or call Trevor at (951) 805-5410 (cell).

Single-cell cDNA sequencing on Revio

Single-cell cDNA requires a minimum of 15 ng with a minimum concentration of 1 ng/uL. Submitting 100 ng is recommended when possible.

Current UCSD Pricing:

Kinnex single-cell RNA library preparation from cells/nuclei - $2700 per sample. This includes 10x Genomics reagents.
Kinnex single-cell RNA library preparation from unamplified barcoded single-cell cDNA - $900 per sample. Generates Single-Cell Isoform cDNA

PacBio Revio sample submission

We accept RNA, DNA, cDNA, cells and tissue for library preparation. We also accept already-made libraries and library pools for sequencing.

To submit your samples,

Email the Revio submission form to gcore@ucsd.edu
Schedule the drop-off time here
Drop off the samples here or mail them to: Trevor Biddle, SCRM, 2880 Torrey Pines Scenic Dr, Rm. B007, La Jolla, CA 92037

Genomic DNA samples should be of high molecular weight, with a minimum of 50% being >30 kb in length. Smaller genomes can be prepared but will have lower yields.
2-5 ug of DNA is required for library preparation. Less than 2 ug can be used, but it is not guaranteed that there will be enough to run a full SMRT cell.

UCSD pricing for DNA

Sequencing - $1600 per SMRT cell (to run a library or a pool of libraries). A typical yield is 60-90 Gbps per SMRT cell for genomic DNA or ~40 million full-length cDNA reads.
1-4 SMRT cells can be run in parallel. Multiple samples can be pooled per a SMRT cell.

Genomic DNA quality control and library preparation - $600 per sample.

UCSD pricing is extended to the University of California, Salk Institute for Biological Studies, Sanford-Burnham Prebys, Scripps Research, and La Jolla Institute for Immunology. Pricing is subject to change based on market conditions and available resources. Please inquire about the external pricing.

DNA extraction for Revio

We accept cells and tissues for DNA extraction for Revio sequencing. For the spleen samples, submit at least 10 mg of tissue.

The price is $135 per sample.

DNA methylation analysis

Revio file outputs contain DNA methylation data by default. No additional biochemical procedures are needed since PacBio technology identifies 5mC in CpG dinucleotides automatically by default. GCore offers additional bioinformatic analysis to align, extract and summarize methylation scores for each CpG. This procedure is computationally intensive and requires a lot of disk space.

Bioinformatic DNA methylation analysis: $160 per sample.

Combined price per sample for DNA sequencing on Revio

Item 20x 30x

---------------------------------------------

HMW DNA extraction $135 $135

QC and library prep $600 $600

Sequencing $1,600 $2,400

Bioinformatic DNA methylation $160 $160

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Total $2,495 $3,295

Summary

The Sanford Consortium Genomics Core is now accepting sample submissions for library preparation and/or sequencing on the PacBio Revio long-read sequencer. This sequencer is proficient in detecting structural variants, haplotypes, methylation, CRISPR mutations, viral insertions, targeted sequencing in DNA, and splice isoforms in RNA, which are difficult to accomplish with traditional short-read sequencing platforms.

Our current output is over 60-90 Gbps per SMRT cell, equivalent to over 20x-30x human genome coverage. We have the capacity to run 4 SMRT cells in a span of 30 hours. The sequencer is optimized to handle fragment sizes in the range of 15-20 kb, allowing for the multiplexing of samples or splitting the sample across multiple SMRT cells.

We offer genomic DNA, bulk, and single-cell Isoform RNAseq library preparations for Revio. For genomic DNA analyses, in addition to sequencing Revio automatically outputs methylation data.

Iso-Seq isoform discovery

Revio Applications
Iso-Seq bulk and single-cell RNA sequencing applications include identification and quantification of RNA isoforms and alternative splicing variants, full-length transcripts, new genes and noncoding RNAs, gene fusions, and analysis of tumor heterogeneity.
DNA applications include de-novo genome sequencing, genome assembly, haplotype reconstruction, allele and haplotype phasing, identification of structural variants, CRISPR mutations, and viral insertions and sequencing through highly repetitive regions up to 20 kb long and genome-wide DNA methylation analysis. DNA methylation analysis is useful for stem cell research and regenerative medicine as it identifies differential methylation in CpG islands in gene promoters, which is the key upstream mechanism leading to cell differentiation and is the key signature of cell lineages and types.

PacBio support

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