PacBio Library Prep & Sequencing
We offer PacBio sequencing services on the PacBio Sequel II and Revio. Each Sequel II SMRTcell contains 8M clusters, and a successful run typically yields around 5M reads. This translates to roughly 20 Gbp of data for circular HiFi reads. However, sequencing output varies significantly depending on library type and quality. Each Revio SMRTcell contains 25M clusters, and a successful run typically yields around 20M reads. This translates to 60-90 Gbp of data for circular HiFi reads.
|Instrument||Clusters||Typical Read Output||Typical Raw Data||Typical Unique Data (CCS)|
|Sequel II||8 Million||5 Million||80 Gbp||20 Gbp|
|Revio||25 Million||20 Million||240 Gbp||80 Gbp|
We can take samples/libraries from any stage and carry them through to sequencing. We offer High Molecular Weight (HMW) extractions, shearing, BluePippin and SageELF size selection, as well as library preps for amplicons, gDNA or mRNA, including low input library preps. Each library prep reaction can produce enough library for 1-3 SMRTcells, depending on input amounts and efficiency. We now also offer full-length sequencing of 10X Genomics single-cell RNA-seq libraries via the MAS-seq kit and protocol.
Circular Consensus Sequencing (CCS) produces reads from 10kb – 20kb in length, and after consensus generation and filtering can produce HiFi reads with 99.9% accuracy.
We can demultiplex, generate CCS/HiFi reads, determine isoforms with the PacBio IsoSeq pipeline, and process read kinetics for subsequent determination of epigenetic modifications. Further analyses cannot be accommodated in house and will be outsourced to UC Davis.
Submitting Tissue Samples
Please contact us at firstname.lastname@example.org before submitting tissue samples.
Nucleic Acid Sample Requirements
Please elute in an EDTA-free buffer.
Quantify using an accurate flourimetric method (e.g. Qubit). If you cannot provide quantification or sizing information prior to submitting we can provide this at an additional cost.
We can work with a little as 100 ng, however we will need to amplify your DNA and there may be an upcharge for using the low or ultra low input kits. Please get in touch with us if you will be submitting less than 3 ug of DNA.
The input amount needed is dependent on the size of your amplicon. Please refer to the chart below. We recommend erring on the side of more material as we see a decent amount of loss during the library prep process.
|Amplicon Size||Input DNA Amounts (per SMRTcell)|
|250-500 bp||250 – 500ng*|
|500-1000 bp||250 – 500ng*|
|1-3 kb||500 – 1000ng|
|3-10 kb||1000 – 2000ng|
|15 kb||1500 – 3000ng|
A high input concentration (>45ng/uL) is necessary for a good prep – the library prep process requires at least 300 ng of RNA in 7 uL.
DNAse treat your sample using an RNAse-free DNAse that does not require heat inactivation.
|Sample Type||Amount (per SMRTcell)||Volume||Sizing||A260/280||A260/230|
|gDNA (HiFi)||5 μg||50 μL||>40kb mode||1.8-2.0||>2.0|
|gDNA (CLR)||5 μg||50 μL||As large as possible|
|Amplicons or Linearized Plasmids||See table below||≥100ng/μL||Please let us know your expected product size|
|RNA||≥1 μg||~20 μL (≥45ng/μL)||RIN >7 (≥8 ideal)||~2.0 or greater||>2.0|
|Single Cell Sequencing via Mas-Seq*||≥1 ng of 10X Chromium 3’ single cell cDNA**||~15 μL (1-5 ng/μL)|
*Please contact us at email@example.com prior to submitting samples for Mas-Seq prep and sequencing.
**We also offer 10X single cell services in house, please contact Justin for more information.
Submitting Prepped libraries
Please contact us at firstname.lastname@example.org to determine how much of the library we will need.