PacBio Library Preparation and Sequencing

The rates for our PacBio Library Prep and Sequencing Services can be found here.

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 ZMWs but the output of a run varies based on application. They types of libraries run on a Revio are very different that the older CLR libraries of the Sequel and dont translate into millions of reads per run. A good HiFi library in the 13-20kb range made from intact, clean HMW gDNA (WGS) should translate to 60-100 Gbp of CCS data from one Revio SMRTcell.

Instrument	ZMWs	Typical Read Output	Typical Raw Data	Typical Unique Data (CCS)
Sequel II	8 Million	5 Million	80 Gbp	20 Gbp
Revio	25 Million	Varies by application	n/a	~60-100 Gbp

We can take samples/libraries from any stage and carry them through to sequencing. There is very little we can’t offer in the world of custom work, starting at any point in the process. Even just providing advice.

We offer High Molecular Weight (HMW) extractions, Femto Prep HMW gDNA visualization, shearing on a Megaruptor 3, all SAGE size selection services, as well as library preps from amplicons, gDNA, total RNA, mRNA, and single cells using all available PacBio kits, Kinnex Arrays, customized Kinnex arrays, custom workflows and also offer low input library preps.

Please refer to the PacBio webpage for more information on all applications available on the Sequel II and Revio.

Submitting Tissue Samples

Please contact us at pacbio@berkeley.edu if you are interested in high molecular weight DNA extractions.

Please email Justin jygchoi@berkeley.edu if you are interested in our 10X services for the single cell kinnex workflow.

Submitting Nucleic Acids for Library Prep

Please provide your samples in 2 aliquots in 1.5 mL DNA Lo-Bind Eppendorf tubes or similar for all PacBio submissions.

DNA: 5-10 μg High Molecular Weight gDNA in 50 μL of an EDTA free elution buffer or Molecular Biology Grade water (RNase/DNase Free)
Amplicon: 1-5 μg amplicon pool in 50 μL of an EDTA free elution buffer or Molecular Biology Grade water (RNase/DNase Free)
RNA: 400-1000 ng Total RNA at a minimum concentration of 45 ng/μL (need 300 ng minimum) in an EDTA free elution buffer or Molecular Biology Grade water (RNase/DNase Free)

AND

All Sample Types: 1:10 QC aliquot in an EDTA free elution buffer or Molecular Biology Grade water (RNase/DNase Free)

Label tubes on the TOP and sides of the tubes. We strongly recommend the following naming scheme:

Name the sample with the PI initials, your initials, and a unique number (e.g. SMFR001), If multiplexing name each sample that will be pooled by adding a letter to the pool name (e.g. SMFR001A, SMFR001B, etc).

Your samples must be sent frozen on dry ice as we will not proceed immediately with your main sample. We would like to minimize the number of freeze-thaws the sample goes though by working on the QC aliquot and keeping the main sample frozen until we need to begin prep. Additionally, if there are any impurities in your samples the only way to prevent further degradation in your main sample is to keep it at -80C. If it is frozen and we note in your QC aliquot that a sample needs a clean, we can still salvage your extraction. However, if the sample is not stored properly (ie: stored at room temp, 4C, or 20C) and is truly contaminated we will be unlikely to rescue the sample.

If you have just extracted your sample and are bringing it into the lab immediately then bring the QC aliquot on ice but the main aliquot should be frozen in an abundance of caution unless you have otherwise communicated with us. In this event, please communicate as we’d like to proceed with samples as fresh as we can.

Submitting Prepped Sequencing Libraries

Please contact us at pacbio@berkeley.edu to determine how much of the library we will need.

PacBio Sample QC

The lab will perform all necessary QC in house prior to moving forward with prep, however, user submitted QC is encouraged but not relied upon due to freeze-thaws and changes in quality due to transport noted for every PacBio destined sample even over small periods of time. Our goal is to limit freeze-thaw on all sample types (RNA especially) and to catch and clean impurities in HMW extractions as fast as possible.

Do not overlook the most important steps in PacBio, QC and proper sample handling. The initial QC hurdles can be frustrating, but they are worth it for the high quality Revio data. Sometimes the best sample for your project and organism is not always perfect. High-quality long read sequencing is still a high bar and we’re here to help you get over it.

PacBio Workflow

1 – Quality Control performance on your sample. You do not have an option to opt-out.

For RNA – Bioanalyzer to check your RIN score and qubit for concentration/total mass

For Amplicons – Fragment Analyzer or Femto Pulse to check size and qubit for concentration/total mass

For HMW DNA – Femto Pulse to check sizing, Nanodrop for purity, and qubit for concentration/total mass

From this step there will be many successes, failures and redos. We can do your HMW extractions if you can’t (sometimes we can’t due to permitting). But this is the most important step, if we make it past this step for RNA and Amplicons you’re “generally” free and clear.

Passing QC metrics:

For RNA – RIN score 7+ (8+ preferred) and a concentration greater than 45 ng/μL, 300 ng needed for prep

For Amplicons – sizing is what the user expected, minimum required mass > 300 ng for < 3kbp amplicons, > 500 ng for > 3kbp amplicons

For HMW DNA – modal sizing > 40kbp, 260/280 between 1.8 and 2.0, 260/230 > 2.0, mass > 3 μg

2 – For HiFi preps (typically WGS), the next step is a Short Read Eliminator (SRE) cleanup and a test shear using the Megaruptor 3. The SRE step will clean the small fragments (< 10kbp) in your HMW gDNA samples from under the HiFi target window. Following the SRE step we will perform a test shear on a small portion of your sample. We can shear the same sample aliquot more than once so this test will be conservative targeting above the HiFi range. If your sample over shears (the fragment size is smaller than expected) that typically indicates the sample is likely not as pure as we thought. We may need to do additional work on your unsheared sample to remove impurities that can lead to poor sequencing performance and nicked DNA. In this event, we will reach out to confirm what additional work may need to be done, perform the work, and go through the QC process again with the cleaned samples. Otherwise, we will proceed with a full shear and library prep.

3 – Once library prep is complete, the final library Fragment Analyzer traces and library concentrations will be provided before sequencing is performed. Sometime a HMW gDNA sample cannot generate the ideal sized HiFi library and the user must decide whether to proceed or not.