Template Preparation and Multiplexing Kits

The TPK 2.0 Bundle contains one kit of each of the following: SMRTbell Express Template Prep Kit (TPK) 2.0 (PN: 101-843-100), SMRTbell Enzyme Clean Up Kit (PN: 101-746-400) and Sequencing Primer v2 (PN: 101-847-900).

The TPK 2.0 bundle builds off of the SMRTbell Express TPK 2.0 workflow—a single-tube, addition-only strategy to minimize handling-induced DNA damage for quick library generation. The SMRTbell Enzyme Clean Up kit helps maximize intact SMRTbell templates. Library generation can be completed as few as 5 hours, excluding size selection. SMRTbell templates can be used to generate large-insert HiFi reads for variant detection or de novo assembly.

Each kit contains sufficient reagents to prepare 9 HiFi libraries using 10 ug of gDNA per preparation.

Note: Sequel and Sequel II System compatible. Kit components are sold separately.

• Resource: Procedure & Checklist - Preparing HiFi SMRTbell Libraries Using SMRTbell Express Template Prep Kit 2.0
• Resource: Procedure & Checklist - Using AMPure PB Bead for Size Selection
• Resource: Procedure & Checklist – Preparing 10 kb Library Using SMRTbell Express Template Prep Kit 2.0 for Metagenomic Shotgun Sequencing
• Product insert: SMRTbell Express Template Prep Kit 2.0
• Product insert: SMRTbell Enzyme Clean Up Kit
• Product insert: Sequencing Primer v2
• SDS: SMRTbell Express Template Prep Kit 2.0
• SDS: SMRTbell Enzyme Clean Up Kit
• SDS: Sequencing Primer v2

The SMRTbell Express Template Prep Kit 2.0 (TPK 2.0) supports insert sizes from 250 bp to >50 kb. This kit employs a single-tube, addition-only strategy to minimize handling-induced DNA damage for library generation in as little as 4 hours, excluding size selection. We recommend this kit for most applications except for shotgun metagenomics (use TPK 1.0, PN: 100-259-100) or large-insert HiFi reads are required.

To generate large-insert HiFi reads, the SMRTbell Enzyme Clean Up Kit (PN: 101-746-400) and Sequencing Primer v2 (PN: 101-847-900) are also needed which can be purchased together in the TPK 2.0 Bundle (PN: 101-843-100).

Each kit contains sufficient reagents to prepare either 18 standard reaction large-insert libraries, 18 Iso-Seq libraries, 48 multiplexed microbial genomes, or 96 reactions for amplicon sequencing.

Note: Sequel and Sequel II Systems compatible.

• Resource: Product Note: Large-insert genomic libraries with SMRTbell Express Template Prep Kit 2.0
• Resource: Product Note: Multiplexed Microbial Genome Sequencing with SMRTbell Express Template Prep Kit 2.0
• Resource: Procedure & Checklist - Preparing gDNA Libraries Using the SMRTbell Express Template Preparation Kit 2.0
• Resource: Procedure & Checklist – Preparing SMRTbell Libraries Using Express Template Prep Kit 2.0 With Low DNA Input
• Product insert: SMRTbell Express Template Prep Kit 2.0
• SDS: SMRTbell Express Template Prep Kit 2.0

The Iso-Seq Express Oligo Kit contains two oligonucleotides to support the Iso Seq Express workflow used in conjunction with the SMRTbell Express Template Prep Kit 2.0 and NEBNext Single Cell/Low Input RNA Library Prep Kit. The oligonucleotides are the PacBio Template Switching Oligo (TSO) and PacBio cDNA PCR primer.

Reagents support 24 RNA transcriptome library preparation.

Note: Sequel and Sequel II Systems compatible.

• Resource: Procedure & Checklist: Iso-Seq Express Template Preparation for Sequel and Sequel II Systems
• Product insert: Iso-Seq Express Oligo Kit
• SDS: Iso-Seq Express Oligo Kit

No-Amp Accessory Kit contains Adapter Annealing Buffer, Sequencing Primer v4, and 10X Primer Buffer v2. Annealing buffer is to be used during SMRTbell Adapter hairpin annealing. Sequencing Primer v4 and 10X Primer Buffer v2 is used to anneal Sequencing Primer v4 to final SMRTbell templates.

Reagent quantities support up to 20 SMRTbell Express TPK 2.0 library preparations using standard input amounts.

Note: Sequel and Sequel II Systems compatible. Pacific Biosciences does not sell a kit for carrying out the overall No-Amp Targeted Sequencing method. Use of this method may require rights to third-party owned intellectual property.

• Resource: Procedure & Checklist – No-Amp Targeted Sequencing Utilizing the CRISPR Cas9 System
• Product insert: No-Amp Accessory Kit
• SDS: No-Amp Accessory Kit

AMPure PB beads are used for 1) DNA cleanup and purification during various stages in the SMRTbell library template preparation workflow, 2) buffer exchanges, 3) template concentration, 4) clean up of excess polymerase and primer from polymerase-bound SMRTbell complexes, and and 5) size selection.

Reagent quantity supplied at 5 mL volume.

Note: Compatible with all PacBio Systems

• Resource: Procedure & Checklist – AMPure PB Bead Purification of Polymerase Bound SMRTbell Complexes
• Resource: Procedure & Checklist - Using AMPure PB Bead for Size Selection
• Product insert: AMPure PB
• SDS: AMPure PB

The Elution Buffer (50 mL) is used with the SMRTbell library template preparation and barcoding associated kits for workflows requiring additional elution buffer and AMPure clean-up steps.

Reagent quantity supplied at 50 mL volume.

Note: Compatible with all PacBio Systems

• Product insert: Elution Buffer (50 mL)
• SDS: Elution Buffer (50 mL)

The Barcoded Overhang Adapter Kit–8A/8B are used in conjuntion with the SMRTbell Express Template Prep Kit 2.0 (PN 100-938-900) for multiplexed microbial multiplexing sequencing. The kits can be combined to perform up to 16-plex of pooled microbial genomes. For the Sequel System, we recommend pooling up to 30 MB of total microbial genomes per SMRT Cell to obtain highly contiguous microbial genome assemblies, and up to 40 MB for advanced users. For Sequel II Systems, up to 48 microbial genomes can be pooled together per SMRT Cell 8M using the 16 barcodes from this kit and/or supplementing with customer oligos from any oligo synthesis provider.

The specific barcodes contained in this kit are as follows for the respective kits:

8A: bc1001, bc1002, bc1003, bc1008, bc1009, bc1010, bc1011, bc1012.
8B: bc1015, bc1016, bc1017, bc1018, bc1019, bc1020, bc1021, bc1022.

Each kit contains 8 barcoded overhang adapters with sufficient reagents to perform 6 ligation reactions for each barcode to support multiplexed microbial genome sequencing, or 12 reactions for amplicon sequencing.

Note: Compatible with all Sequel and Sequel II Systems

• Resource: Procedure & Checklist - Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0
• Resource: Express Microbial Multiplexing Calculator
• Resource: Application Note: Microbial Multiplexing Workflow on the Sequel System using SMRTbell Express Template Prep Kit 2.0
• Resource: Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Overhang Adapters for Multiplexing Amplicons
• Product insert: Barcoded Overhang Adapter Kit 8A
• Product insert: Barcoded Overhang Adapter Kit 8B
• SDS: Barcoded Overhang Adapter Kit 8A
• SDS: Barcoded Overhang Adapter Kit 8B

Barcoded Universal F/R Primers Plate–96 v2 is a 96-well plate containing barcoded primers. Each well on the plate contains a primer pair consisting of forward and reverse primers having the same barcode. Each barcode is present in only one well for a total of 96 different barcodes on the plate. Each primer has a universal sequence on the 3' end to allow for incorporation of barcodes into templates through a two-step PCR strategy. The Barcoded Universal F/R Primers Plate - 96 v2 can be used with SMRTbell Template Prep Kit 1.0 or Express Template Prep Kit 2.0.

Reagent quantities support a single use of each of the 96 primers pairs in library preparations.

Note: Compatible with all PacBio Systems

• Resource: Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplexing Amplicons
• Product insert: Barcoded Universal F/R Primers Plate–96 v2
• SDS: Barcoded Universal F/R Primers Plate–96 v2

The SMRTbell Barcoded Adapter Complete Prep Kit–96 includes the SMRTbell Barcoded Adapter Prep Kit (PN: 100-465-800) and Barcoded Adapter Plate – 96 (PN: 100-466-000). These adapters are ligated directly to amplicons and pooled for damage repair using the SMRTbell Damage Repair Kit - SPv3 (PN:100-992-200, sold separately) prior to sequencing. This kit is based on the SMRTbell Template Prep Kit 1.0 workflow.

For Sequel II Systems, suggested barcode designs for barcoded adapters which are compatible with the SMRTbell Express Template Preparation Kit 2.0 are available and can be ordered from any customer oligo provider.

Reagent quantities support a single use of each of the 96 primers pairs in library preparations.

Note: Compatible with Sequel System Kit components also sold separately.

• Resource: Procedure & Checklist - Amplicon Template Preparation and Sequencing
• Product insert: SMRTbell Barcoded Adapter Prep Kit
• Product insert: Barcoded Adapter Plate – 96
• SDS: SMRTbell Barcoded Adapter Prep Kit
• SDS: Barcoded Adapter Plate – 96

The SMRTbell Damage Repair Kit-SP v3 contains reagent supplies to perform DNA Damage Repair, Exonuclease III and VII treatments, and primer annealing of SMRTbell libraries made with SMRTbell Template Prep Kit 1.0 using Sequencing primer v3 for insert sizes ranging from 500 bp to over 20 kb. Recommended for use with the SMRTbell Barcoded Adapter Complete Prep Kit (PN: 100-514-900) to generate primer-annealed SMRTbell libraries ready for polymerase binding. Can also be used for extra DNA damage repair of template.

Reagent quantities support 10 library preparations.

Note: Compatible with Sequel SystemSequencing Primer v3 (PN: 100-970-100) also sold separately.

• Resource: Procedure & Checklist - Amplicon Template Preparation and Sequencing
• Product insert: SMRTbell Damage Repair Kit
• Product insert: Sequencing Primer v3
• SDS: SMRTbell Damage Repair Kit
• SDS: Sequencing Primer v3