We offer PacBio sequencing services on the PacBio Sequel II. Each SMRTcell contains 8M clusters, and a successful run typically yields around 5M reads. This translates to roughly 80 Gbp of data for Continuous Long Read libraries, and 20 Gbp for circular HiFi reads. However, sequencing output varies significantly depending on library type and quality.
We can take samples/libraries from any stage and carry them through to sequencing. We offer High Molecular Weight (HMW) extractions, PCI cleans for user provided gDNA, shearing, BluePippin and ELF size selection, and of course 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.
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. Continuous Long Read (CLR) sequencing can produce single pass reads exceeding 100kb, but have a much higher error rate, particularly around homopolymers.
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.
|Sample Type||Amount (per SMRTcell)||Volume||Sizing||A260/280||A260/230|
|gDNA (HiFi)||5 ug||50 uL||>40kb mode||1.8-2.0||>2.0|
|gDNA (CLR)||5 ug||50 uL||As large as possible|
|Amplicons or Linearized Plasmids||See table below||≥100ng/uL||Please let us know your expected product size|
|RNA||≥1ug||~20 uL (≥45ng/uL)||RIN >7 (≥8.5 ideal)||~2.0 or greater||>2.0|
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.
Submitting Prepped libraries
Please contact us at email@example.com to determine how much of the library we will need.