www.pacb.com/isoseq HUMAN BIOMEDICAL RESEARCH PLANT AND ANIMAL SCIENCES Non-size Selected Iso-Seq Libraries Full-length Transcript Size Non-Size- Selected SMRTbell Library PacBio RS II Sequel System Depicted on the left is a histogram plot of number of full-length sequences by transcript length for a Magbead-loaded, non-size selected Iso-Seq library sequenced on both the PacBio RS II and the Sequel System. The full-length cDNA sequences run on the Sequel System closely resemble the size distribution of the input SMRTbell library (shown on the right). FROM RNA TO ACCURATE GENE MODELS LONG-READ RNA SEQUENCING BEST PRACTICES With Single Molecule, Real-Time (SMRT ® ) Sequencing and the Sequel ® System, you can easily and affordably sequence transcript isoforms of up to 10 kb in their entirety. The Iso-Seq ® method allows users to generate full-length cDNA sequences - with no assembly required - in order to confidently characterize the full complement of transcript isoforms within targeted genes, or across an entire transcriptome. SAMPLE PREPARATION RECOMMENDATIONS - Prepare full-length transcripts using the Clontech® SMARTer® PCR cDNA Synthesis Kit with as little as 1 ng of poly-A+ RNA or 2 ng of total RNA 1 - The Sequel Sequencing Kit and protocols eliminate the need for size selection for transcripts <4 kb 2 - Optional size-selection protocols to enrich for transcripts >4 kb - Compatible with standard target enrichment methods, such as NimbleGen SeqCap EZ 3 or IDT xGen Lockdown Probes 4 - Multiplex with sample barcoding 5 - Scalable throughput - Up to 20 Gb, or 250k-350k full-length non-chimeric reads, per SMRT Cell 1M* - Profile transcripts from multiplexed samples in a single SMRT Cell - Survey transcriptomes in 1–2 SMRT Cells on the Sequel System - Increase sequencing depth for more comprehensive transcriptome characterization MORE CONSISTENT LOADING ON SEQUEL SYSTEM REDUCES NEED FOR SIZE SELECTION Analyze with SMRT Analysis Software Suite poly-A+ RNA Total RNA Optional Poly-A Selection Reverse Transcription Full Length 1 st Strand cDNA Large-scale Amplification Amplified cDNA >4 kb Combined SMRTbell Library SMRT Sequencing on Sequel System Optional Size Selection * Read lengths, number of reads, data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size, among other factors.
2
Embed
Read full-length transcripts – no assembly required (2015)
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
w w w. p a c b .c o m / i s o s e q
HUMAN BIOMEDICAL RESEARCH
PLANT AND ANIMAL SCIENCES
Non-size SelectedIso-Seq Libraries
Full-length Transcript Size
Non-Size-Selected SMRTbell
Library
PacBio RS IISequel System
Depicted on the left is a histogram plot of number of full-length sequences by transcript length for a Magbead-loaded, non-size selected Iso-Seq library sequenced on both the PacBio RS II and the Sequel System. The full-length cDNA sequences run on the Sequel System closely resemble the size distribution of the input SMRTbell library (shown on the right).
FROM RNA TO ACCURATE GENE MODELS
LONG-READ RNA SEQUENCING BEST PRACTICES
With Single Molecule, Real-Time (SMRT®) Sequencing and the Sequel® System, you can easily and affordably sequence transcript isoforms of up to 10 kb in their entirety. The Iso-Seq® method allows users to generate full-length cDNA sequences - with no assembly required - in order to confidently characterize the full complement of transcript isoforms within targeted genes, or across an entire transcriptome.
SAMPLE PREPARATION RECOMMENDATIONS - Prepare full-length transcripts using the Clontech®
SMARTer® PCR cDNA Synthesis Kit with as little as 1 ng of poly-A+ RNA or 2 ng of total RNA1
- The Sequel Sequencing Kit and protocols eliminate the need for size selection for transcripts <4 kb2
- Optional size-selection protocols to enrich for transcripts >4 kb
- Compatible with standard target enrichment methods, such as NimbleGen SeqCap EZ3 or IDT xGen Lockdown Probes4
- Multiplex with sample barcoding5
- Scalable throughput - Up to 20 Gb, or 250k-350k full-length non-chimeric
reads, per SMRT Cell 1M* - Profile transcripts from multiplexed samples in a
single SMRT Cell - Survey transcriptomes in 1–2 SMRT Cells on the
Sequel System - Increase sequencing depth for more comprehensive
transcriptome characterization
MORE CONSISTENT LOADING ON SEQUEL SYSTEM REDUCES NEED FOR SIZE SELECTION
Analyze with SMRT Analysis Software Suite
poly-A+ RNA
Total RNA
Optional Poly-A Selection
Reverse Transcription
Full Length1st Strand cDNA
Large-scale Amplification
Amplified cDNA >4 kb
Combined SMRTbell Library
SMRT Sequencing on Sequel System
OptionalSize Selection
* Read lengths, number of reads, data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size, among other factors.
KEY REFERENCES1. Procedure & Checklist – Iso-Seq™ Template Preparation for Sequel® Systems2. Clark, T. et al. (2017) Full-Length cDNA Sequencing on the PacBio Sequel Platform. Poster presented at Plant and Animal Genome Conference. San
Diego, CA.3. Full-length cDNA Target Sequence Capture Using SeqCap® EZ Libraries4. Full-length cDNA Target Sequence Capture Using IDT xGen® Lockdown® Probes5. Barcoding Samples for Isoform Sequencing (Iso-Seq Analysis)6. Best practice for analyzing multiplexed Iso-Seq data7. PacBio Support: Software Downloads8. Running SMRT Analysis on Amazon 9. Tutorial: Iso-Seq Analysis Application10. Abdel-Ghany, S.E. et al. (2016) A survey of the sorghum transcriptome using single-molecule long reads. Nature Communications. 7, e11706.
The Iso-Seq method allows you to make evidence-based genome annotations, discover novel genes and isoforms, identify promoters and splice sites to understand gene regulation, improve accuracy of RNA-seq quantification for gene expression studies, and distinguish important stress response, developmental, or tissue-specific isoforms.
Splice isoform analysis in the sorghum transcriptome using the Iso-Seq method greatly improved genome annotation, with >11,000 novel splice isoforms and >2,100 novel genes identified. In this example, a gene was discovered to produce 13 novel alternatively spliced isoforms, where the previous gene model contained only a single isoform10.
The Iso-Seq protocol, available in SMRT Analysis, generates consensus sequences and determines those transcripts that are full-length by detecting and identifying the 5’ primer, Poly-A sequence, and the 3’ primer of the reads.
INFORMATICS PIPELINE FOR ISO-SEQ ANALYSIS
DATA ANALYSIS SOLUTIONS WITH PACBIO SMRT ANALYSIS - Use the Iso-Seq protocol in SMRT Analysis to output high quality, full-length transcript sequences, with no assembly
required, to characterize transcripts and splice variants and map transcripts back to a reference genome - ‘IsoSeq’ option recommended for analysis of targeted Iso-Seq experiments; ‘IsoSeq2 (beta)’ recommended for whole
transcriptome analysis6
- Run Iso-Seq analysis in either de novo (no genome reference required) or reference-based mode - Install SMRT Analysis locally7 or access it via Amazon Cloud8
- View tutorial9 for running the Iso-Seq protocol in SMRT Analysis