PacBio Single Molecule Sequencing

Helpful Links Related to PacBio Sample Preparation

PacBio Nanobind HMW DNA extraction overview

Technical-Note-Preparing-DNA-for-PacBio-HiFi-Sequencing-Extraction-and-Quality-Control

DNA Extraction Protocols

High-quality, high-molecular-weight genomic DNA is crucial for obtaining long read lengths and optimal sequencing performance. The SMRT library preparation process does not utilize amplification techniques and resulting library molecules are used directly as templates for the sequencing process. The quality of the DNA and RNA starting material will directly impact the extent of sequencing success or failure. Any unrepaired or irreversible DNA damage present in the input material (e.g., interstrand crosslinks, nicks, etc.) or contaminants will result in impaired performance in the system.

Upon receiving your DNA sample, a QC of Qubit, Nanodrop, and Fragment Analyzer is performed to ensure the DNA meets our concentration, purity, and size requirements, respectively.

Ideally, the 260/230 should be within the 2.0-2.2 range, and the 260/280 within 1.8-2.0 range. Initial DNA/RNA quality is critical for a successful run.

If you are submitting 24 or more samples, please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.
PLEASE USE V BOTTOM PLATES DESIGNED FOR MOLECULAR BIOLOGY AND NOT FLAT BOTTOM PLATES DESIGNED FOR OTHER PURPOSES. 

If your samples do not meet the requirements in the table below, please contact us to discuss potential options for processing your project.

 

PacBio Library Prep

Application	Library Type	Description	Multiplexing	Mass Requirement	Volume Requirement	Other Requirements
Whole Genome Sequencing	De Novo Assembly - HiFi Reads	Produce reference quality assemblies for genomes	_	1 ug/ Gb of genome	≥50 uL	50% ≥ 30 kb & 90% ≥ 10 kb
	Variant Detection	With 2 SMRT Cells 8M, Call SNVs, InDels, and SVs in a 3 Gb genome	_	1 ug/ Gb of genome	≥50 uL	50% ≥ 30 kb & 90% ≥ 10 kb
	De Novo Assembly - for Low DNA Input	Produce reference quality assemblies for genomes up to 1 Gb. Multiplex up to 2 small genomes on the Sequel II System	two 600-Mb genomes	>400 ng per 1 Gb genome size (single-sample), >300 ng per 600 mb genome size (2-plex)	≥50 uL	50% ≥ 30 kb & 90% ≥ 10 kb
	Microbial De Novo Assembly	Sequence up to 96 microbes	up to 96 microbes	>300 ng per microbe	≥50 uL	90% ≥ 7 kb
	De Novo Assembly and Variant Detection - for UltraLow DNA Input	Produce reference quality assemblies for genomes up to 500 Mb	_	5-20 ng per 500 Mb genome size	≥50 uL	Majority of gDNA >20 kb
Viral Sequencing	HiFiViral SARS-CoV-2	This procedure captures the SARSCoV2 genome with tiled molecular inversion probes that create overlapping amplicons resulting in comprehensive sequence coverage	up to 384 SARS-CoV-2 samples	>10,000 copies of SARS-CoV-2 RNA per sample	≥10 uL	_
	Adeno-associated virus (AAV)	Generate long and accurateHiFi reads, which can sequence an AAV genome from ITR to ITR, revealing otherwise difficult-to-detect issues	up to 24 AAV samples	≥ 1 µg of AAV DNA per SMRT Cell, or per sample amounts should be 1 µg/ number of samples	≥50 uL	_
RNA Sequencing	Iso-Seq Method	Characterize alternative splicing/annotate a genome with full length transcripts	up to 12 Iso-Seq samples/SMRT Cell 8M for genome annotation	300 ng total RNA	≥10 uL	RIN (RNA integrity number) ≥7.0
	MAS-Seq for 10x Single Cell	Identify cell type-specific isoforms and take advantage of long and accurate HiFi read lengths to increase single-cell Iso-Seq throughput by 16-fold through concatenated arrays of single-cell cDNA molecules	_	15 ng per library or 60-75 ng per library	≥15 uL	Optimal range of 3,000-10,000 target cell recovery from the 10x Chromium 3’ single cell workflow
Metagenomics	Full-length 16S rRNA Sequencing	Multiplex up to 192 samples to provide strain level resolution	up to 192 samples	1000 ng of pooled 16S samples	≥50 uL for a pool	_
	Shotgun Metagenomic Profiling or Assembly	Generate near-complette assemblies of high-complexity sample(s) (e.g. gut microbiome)	profile up to 48 communities, assemble up to 4 communities	>300 ng per sample	≥50 uL	90% ≥ 7 kb
Targeted Sequencing	Amplicon Sequencing	HiFi sequencing can span your amplicon in a single read, giving unambiguous haplotype resolution through direct phasing	up to 1,000+ samples	Total input DNA per SMRT Cell 8M: 300 ng for <3 kb, 500 ng for 3 - 10 kb, ≥1000 ng for ≥10 kb	≥50 uL for a pool	_

Contact for Financial Inquiries and Quote Requests

Please email Kim and Elizabeth at ggbc@uga.edu, for financial inquiries or to request a quote. Be as specific as possible, so that they can more quickly assist you.

A Note About Single SMRTcell Projects:

SMRTcell sequencing results depend on the final loading concentration of the library. Due to the nature of the technology, optimal concentrations can vary greatly between libraries and PacBio recommends running 2 titration SMRTcells to find the optimal concentration before running production SMRTcells. With this in mind, please be aware that projects requesting a single SMRTcell may have variable results. In most cases the first SMRTcell performs well and this is not an issue, but sometimes additional sequencing is needed. When this happens, the customer is expected to cover the cost of additional sequencing.

PacBio Table

PacBio Service	Description	UGA Fee	Non-UGA Fee	Commercial Fee
HMW DNA or RNA Extraction
DNA or RNA QC prior to extraction/purification	Volume, Concentration (ng/ul) and DNA Size	Inquire	Inquire	Inquire
HMW DNA extraction from Plant leaf tissue	High molecular Weight DNA extraction suitable for HiFi library preparation	Inquire	Inquire	Inquire
RNA preparation	RNA preparation from frozen tissue	Inquire	Inquire	Inquire
Library Preparation
CCExpress Library (HiFi) Indexed	10 µg HMW DNA minimum required	Inquire	Inquire	Inquire
IsoSeq Express	Express IsoSeq library sized with beads	Inquire	Inquire	Inquire
Sequencing
Revio 24-h movie time	IsoSeq and short amplicons not currently supported	Inquire	Inquire	Inquire
Sequel II 30-h movie time	IsoSeq and short amplicons are supported	Inquire	Inquire	Inquire
Data Analysis
Data Analysis	See https://dna.uga.edu/bioinformatics	Inquire	Inquire	Inquire

The standard output files from the PacBio sequencing run are: 

Raw subreads	CCS (HiFi) reads
*.baz2bam_1.log	ccs_processing.report.json
*.scraps.bam	ccs.report.csv.zip
*.scraps.bam.pbi	ccs.report.json
*.sts.xml	ccs_tasks_report.json
*.subreads.bam	ccs_zmws.json.gz
*.subreads.bam.pbi	final.consensusreadset.xml
*.subreadset.xml	*.hifi_reads.bam
*.transferdone	*.hifi_reads.fasta.gz
tmp-file*.txt	*.hifi_reads.fastq.gz
	reads.bam

 

We will be sending you the following files: 

Raw subreads	CCS (HiFi) reads
*.baz2bam_1.log	ccs.report.csv.zip
*.sts.xml	final.consensusreadset.xml
*.subreads.bam	*.hifi_reads.bam
*.subreads.bam.pbi	*.hifi_reads.fasta.gz
	*.hifi_reads.fastq.gz
	reads.bam

 

For multiplexed runs, you will be receiving both raw and demultiplexed data.

GGBC uses Globus to transfer the data. We ask that you please confirm when you received and downloaded the data. Once your download link expires, your data is deleted on our end so please contact us beforehand if you have issues downloading the data.

1. Whole Genome Sequencing:
   1. Variant Detection
   2. De Novo Sequencing
   3. Genome Structure
   4. Epigenetics
2. Targeted Sequencing:
   1. Human Genomics
   2. HLA Sequencing
   3. Plant + Animal Sciences
   4. Microbial + Infectious Disease Research
3. RNA Sequencing:
   1. Human Transcriptome Studies
   2. Plant + Animal Transcriptome Studies
   3. Single-Cell Transcriptome Studies
4. Epigenetics Sequencing:
   1. Human and Eukaryote Epigenetics
   2. Microbial Epigenetics
5. Metagenome Sequencing