PacBio

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 gsl-fgl@berkeley.edu 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.

DNA

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.

Amplicons

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

RNA

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 gsl-fgl@berkeley.edu 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 gsl-fgl@berkeley.edu to determine how much of the library we will need.