Long-read Sequencing & Library Construction

Guidelines for Sample Input

• The amounts in tables are the minimum sheared DNA needed for input into SMRTbell template prep.
• Please provide minimum 5ug of genomic DNA per sample to have ample sample for initial QC and shearing.
• After Corvaris shearing there is a beads clean up and there can be variable loss depending on the quality of the gDNA samples so it is helpful to have additional sample where possible.
  

  DNA input	Revio™
  Single or pooled library	2 µg per Revio SMRT Cell
  Multiplex libraries	300 ng – 2 µg per sample
  DNA Quality	Human, plant, and animal samples	Microbial and Metagenomic samples
  DNA size distribution	50% ≥30 kb & 90% ≥10 kb	90% ≥7 kb

DNA Quality

• For human, animal, and plant genomes, the recommended Genome Quality Number (GQN) is 9.0 or higher at 10 kb and 5.0 or higher at 30 kb. High-quality genomic DNA will maximize sequencing coverage per SMRTbell library and reduce sequencing costs.
• For samples that do not meet these metrics, the PacBio SRE kit can be used to improve sample quality by depleting degraded DNA prior to shearing.
• For prokaryotic genomes or metagenomic sequencing applications, the recommended GQN is 9.0 or higher at 7 kb. Samples should be free of RNA before beginning library prep. If RNA is present, treat with RNase A and incubate at 37C for 15 minutes. Clean reactions using a 1X concentration of SMRTbell cleanup beads (or AMPure PB) before proceeding. RNA can reduce loading of your library on the SMRT Cell if not removed prior to library prep.

DNA Input Note

• Using lower than recommended DNA input amounts may result in insufficient SMRTbell library product for loading at optimal concentrations.
• Each library prep reaction supports up to 5 µg of sheared genomic DNA. As little as 300 ng of DNA input may be used per sample when multiplexing. However, the sum across all samples should be greater than the recommended minimal DNA amount per SMRT Cell (2 µg for Revio system). For example, if planning to multiplex 4 samples on a Revio SMRT Cell, use at least 500 ng of sheared DNA per sample.
• For complete protocol, see the PacBio Procedure & checklist - Preparing whole genome and metagenome libraries using SMRTbell® prep kit 3.0

Whole genome sequencing

• De novo genome assembly w/ ultralow input: Produce high-quality, haplotype-phased genome assemblies.

HiFi SMRTbell® templates from Ultra-Low DNA Input

• This procedure describes how to prepare HiFi SMRTbell libraries from 20 ng of input genomic DNA (gDNA) for sequencing on the Revio system. Genomic DNA is sheared to approximately 10 kb using a g-TUBE or a Megaruptor system, amplified by PCR, constructed to a SMRTbell library and can be size-selected using the BluePippin system. This workflow enables de novo assembly of insect genomes of up to 500 Mb (>500 Mb genome size is not supported) and human variant detection from as low as 5 ng gDNA.
• Guidelines for sample input (see below)
  

  Required gDNA Input Amount	Required Quality of Input gDNA	gDNA Shearing Method	Target Sheared Fragment Size Distribution Mode	Amplification Target Size Distribution Mode	Total Mass of Pooled PCR Product Required for Library Construction	Required SMRTbell Library Input for BluePippin Size-Selection
  5-20 ng	Majority of gDNA > 20 kb	Megaruptor or g-TUBE	10 kb sheared DNA is optimal	8-10 kb	≥500 ng	≥400 ng

• PacBio recommends using high molecular weight gDNA with majority >20 kb for library construction. A DNA sample containing significant amounts of < than 10kb fragments will result in preferential amplification of short fragments thereby resulting in short reads that are not ideal for de novo assembly and resequencing for variant detection. Therefore, it is critical to understand the quality of your gDNA sample prior to shearing.
• The Femto Pulse system is highly recommended because it requires significantly lower amounts of DNA (200 - 500 picograms) than other systems. Complementary to Femto Pulse, the Qubit High Sensitivity (HS) is highly recommended for measuring DNA concentration. For constructing a SMRTbell library, a minimum 500 ng of amplified sample (PCR reaction 1 + PCR reaction 2) is required to generate sufficient SMRTbell library for 1 Revio SMRT® Cell 8M. To generate 2 Revo? SMRT Cells 8M, we recommend starting with approximately 800 ng of pooled amplified sample for library construction.
• For complete protocol, see the PacBio Procedure & Checklist - Preparing HiFi SMRTbell® Libraries from Ultra-Low DNA Input.
  

RNA sequencing

• Whole transcriptome (Iso-Seq method)
• Applications from PacBio website:
  • Characterize alternative splicing
  • Annotate a genome with full-length transcripts

Iso-Seq® libraries using SMRTbell® prep kit 3.0

• Guidelines for sample input
• This protocol requires high-quality RNA. Prior to library preparation, we need to evaluate the size distribution of the input RNA to determine whether it is suitable for the protocol.

RNA Integrity Number (RIN) > 7

• Please provide 1ug of total RNA so there is enough sample for initial QC (qubit RNA High Sensitivity for quantification and Bioanalyzer Agilent for quality for RIN information).
• If you already ran your total RNA on either TapeStation or Agilent Bioanalyzer, please provide that information to us at the time of your sample submission.
• Minimum input of total RNA is 300ng per sample per SMRTbell template prep
• Using the concentration of cDNA reading from the Qubit fluorometer, pool an equal mass of each barcoded cDNA prep
• cDNA
  • maximum total combined mass without exceeding 500 ng and not less than 100 ng in 46 μL
• Minimum cDNA
  • cDNA input 100 ng in 46 μL
  • maximum total combined mass possible without exceeding 500 ng and not less than 100 ng in 46 μL
• For complete protocol v2, see the PacBio Procedure & Checklist - Preparing Iso-Seq® v2 libraries using SMRTbell® prep kit 3.0
  

Single-Cell Iso-Seq (MAS-Seq)

• Characterize alternative splicing in single cells / cell types
• MAS-Seq SMRTbell templates using MAS-Seq for 10x Single Cell 3’ kit
• This kit is intended for use with single-cell cDNA generated using the 10x Chromium Next GEM Single Cell 3’ kit (v3.1), standard throughput.
• cDNA Input Optimal range of 3,000-10,000 target cell recovery from the 10x Chromium 3’ single cell workflow
• This protocol requires at least 15 ng of 10x Chromium 3’ single cell cDNA. (Input amount 15ng-75ng).
• Follow the 10x Chromium user guide through? cDNA amplification, cleanup and QC (refer to Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 user guide). Before you begin, evaluate the quantity and size distribution of input cDNA to determine whether it is suitable to put into the MAS seq protocol (average size between 500-1500bp).
• For complete protocol, see the PacBio Procedure & Checklist - Preparing MAS-Seq libraries using MAS-Seq for 10x Single Cell 3’ kit 

Targeted Sequencing

• Generate sequences of complete long-range amplicons
• Multiplexed amplicon SMRTbell templates using SMRTbell® prep kit 3.0

SMRTbell adapter index plate 96A- we can plex up to 96 amplicons

• Amplicons can be barcodes prior to SMRT bell template construction using the barcoded M13 primer plate contains 384, 16 bp dual indices
  This procedure describes the workflow for constructing amplicon libraries using the SMRTbell prep kit 3.0 for sequencing on PacBio® Revio", Sequel® Il, and Ile systems. Amplicons may be indexed during PCR, or during library preparation with SMRTbell indexed adapters.

  Overview
  	PCR-indexed samples	Adapter-indexed samples
  Sample per kit	1-24	1-24
  Workflow time	3.5 hours	4 hours
  Size	250-25,000 bp	250-25,000 bp
  DNA input per Revio SMRT® Cell	300-2000 ng per pool	300-2000 ng per pool
  DNA input per Sequel II Revio SMRT® Cell 8M	150-1000 ng per poo	150-1000 ng per sample

• PCR-indexed sample input
  If samples were indexed during PCR, then the samples can be pooled prior to SMRTbell template prep and the total DNA input amount will equal the amount of the multiplexed pool. The per sample input will equal the total DNA input divided by the number of multiplexed samples.
  

  Mean size	Minimum pooled amount per Revio SMRT® Cell
  <3 kb	300 ng
  3-12 kb	500 ng
  >12 kb	700 ng

• Adapter-indexed sample input
  

  Mean size	Minimum per sample amount per Revio SMRT® Cell
  <3 kb	300 ng
  3-12 kb	500 ng
  >12 kb	750 ng

• For complete protocol, see the PacBio  Procedure & Checklist - Preparing multiplexed amplicon libraries using SMRTbell® prep kit 3.0 and the Procedure & Checklist - Preparing multiplexed amplicon libraries using PacBio barcoded M13 primers and SMRTbell® prep kit 3.0