Template Preparation and Multiplexing Kits

The SMRTbell Express Template Prep Kit 2.0 supports insert sizes from 500 bp to >50 kb. This kit employs a single-tube, addition-only strategy to minimize handling-induced DNA damage for library generation in 4 hours. We recommend this kit for large-insert gDNA libraries for de novo whole genome sequencing, and multiplexed microbial genome sequencing. Currently, the kit has not been optimized for preparing templates for Iso-Seq analysis, and is not recommended for this application. The kit is designed for diffusion loading only and is currently only compatible with the Sequel System.

Each kit contains sufficient reagents to prepare 18, 48, and 96 reactions to respectively support large-insert libraries, microbial multiplexing, and amplicon sequencing.

Note: Sequel and Sequel II Systems compatible. Amplicon solutions accompanying this kit will be available in Summer 2019.

• Resource: Procedure & Checklist - Preparing gDNA Libraries Using the SMRTbell Express Template Preparation Kit 2.0
• 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 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 SMRTbell Template Prep Kit 1.0-SPv3 bundle contains the reagents, including sequencing primer, needed to prepare SMRTbell libraries for the Sequel System. This kit is recommended for amplicons, genomic libraries, Iso-Seq analysis, and use with any of the barcoding products for multiplexing.

Reagent quantities support the preparation of 10 SMRTbell libraries. For bulk packaging, please contact your local field application scientist or sales representative.

Note: Sequencing Primer v3 (PN: 100-970-100) also sold separately.

• Resource: Procedure & Checklist: 20 kb Template Preparation Using BluePippin Size-Selection System for Sequel Systems
• Resource: Procedure & Checklist: Preparing Multiplexed Microbial SMRTbell Libraries for the PacBio Sequel System
• Resource: Procedure & Checklist - Amplicon Template Preparation and Sequencing
• Product insert: SMRTbell Template Prep Kit 1.0
• Product insert: Sequencing Primer v3
• SDS: SMRTbell Template Prep Kit 1.0
• SDS: Sequencing Primer v3

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 and 4) clean up of excess polymerase and primer from polymerase-bound SMRTbell complexes.

Reagent quantity supplied at 5 mL volume.

• Resource: Procedure & Checklist – AMPure PB Bead Purification of Polymerase Bound SMRTbell Complexes
• 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.

• 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. 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.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.

• Resource: Procedure & Checklist - Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0
• Resource: Express Microbial Multiplexing Calculator
• Resource: Analysis Procedure - Multplexed Microbial Assembly with SMRT Link v6.0.0 and Express Template Prep Kit 2.0
• Resource: Application Note: Microbial Multiplexing Workflow on the Sequel System using SMRTbell Express Template Prep Kit 2.0
• 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

The Barcoded Universal F/R Primers Plate -96 contains a 96-well plate of barcoded forward and reverse primers. Each well on the plate contains a unique primer pair consisting of a forward and reverse primer of the same barcode. These barcode sequences are incorporated with amplicons via a 2-step PCR process where primer pairs designed to specific targets have also been tagged with a universal sequence compatible with the Barcoded Universal F/R Primers Plate - 96. Subsequently, standard SMRTbell adapters are ligated to these barcoded amplicons.

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

• Resource: Procedure & Checklist - Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing
• Resource: Product Note: Multiplexing Amplicons Up To 10 kb
• Resource: SMRT Analysis Barcoding Overview (v5.1.0)
• Product insert: Barcoded Universal F/R Primers Plate -96
• SDS: Barcoded Universal F/R Primers Plate -96

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.

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

Note: Kit components also sold separately.

• Resource: Procedure & Checklist - Preparing Amplicon Libraries using PacBio Barcoded Adapters for Multiplex SMRT Sequencing
• Resource: Product Note: Multiplexing Amplicons Up To 10 kb
• Resource: SMRT Analysis Barcoding Overview (v5.1.0)
• 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-SPv3 contains reagent supplies to perform DNA Damage Repair, Exonuclease III and VII treatments, and primer annealing of SMRTbell libraries 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: Sequencing Primer v3 (PN: 100-970-100) also sold separately.

• Resource: Procedure & Checklist - Preparing Amplicon Libraries using PacBio Barcoded Adapters for Multiplex SMRT Sequencing
• Product insert: SMRTbell Damage Repair Kit
• Product insert: Sequencing Primer v3
• SDS: SMRTbell Damage Repair Kit
• SDS: Sequencing Primer v3