Output

Thanks to recent advances in sequencing chemistry and adaptive loading, the SMRT Cell 8M typically generates ~ 4-5M raw reads with flexible sequencing run times of up to 30 hours, yielding ~250-450 Gbp of raw data. As the sequencing polymerase makes successive passes along the same SMRT bell template, random errors present in each subread can be corrected to achieve high yields of >QV20 (99%) without sacrificing read length.

• Up to 160 Gb when using the Continuous Long Read (CLR) mode for de novo assembly and structural variant detection. By size selecting library fragments ≥30 kb, exceptionally long reads can be achieved with some > 150 kb. 
• Up to ~500 Gb when using Circular Consensus Sequencing (CSS, HiFi) mode with 10 - 15 Gb after CSS and filtering for highly accurate amplicon and whole transcriptome sequencing projects.

Benefits

• Long Reads: With HiFi read lengths up to 25 kb,  investigators can readily assemble complete genomes and sequence full-length transcripts. Long read lengths can resolve repetitive regions that are difficult for short-read technologies. 
• High Accuracy: >99.999% consensus accuracy is achieved by sequencing the same molecule multiple times.
• Uniform Coverage: No bias based on GC content enables sequencing through regions inaccessible to other technologies. Template preparation and sequencing do not rely on amplification, thus there is no PCR bias for more uniform genome coverage. 
• Epigenetics: With no PCR amplification step, base modifications are directly detected during sequencing without the need for bisulfite conversion. 

Sample requirements

PacBio workflows are sensitive to quality and quantity of input material. It is important to avoid steps in sample extraction and storage that may cause mechanical shearing, fragmentation, or degradation of high molecular weight (HMW) gDNA. Moreover, PacBio is an amplification-free platform, meaning higher input mass is often required for library prep. UMGC performs a full battery of QC on all PacBio submissions. Only samples passing rigorous QC are taken into library preparation. The table below lists guidelines for minimum and recommended sample input:

• Amplicons - 1-10 ng
• Genomic DNA (higher organism) - 5-10 µg
• Genomic DNA (microbial) - 0.5-5 µg
• Full-length (poly-A) mRNA - >300 ng, RIN ≥ 8.0

Questions?

As a PacBio Certified Service Provider with five years of experience with the Sequel Systems, we undergo standardized PacBio certification and speak at national PacBio user group meetings to ensure investigators receive the highest quality data and the longest reads possible. Please contact us at next-gen@umn.edu for comprehensive support on experimental design.