BRING THE “W” BACK TO
WHOLE GENOME SEQUENCING
Single Molecule, Real-Time (SMRT®) Sequencing delivers long reads with uniform coverage for the
most comprehensive de novo genome assemblies generated today. True whole genome sequencing
provides complete and accurate views of all types of genomic variation revealing structural variants,
mobile elements, haplotypes, low complexity regions, and epigenetic modifications.
-- Go beyond draft genomes, generate megabasesized contigs and finished assemblies
p25.3 p24.1 p22.1 p15 p13.1 q11.1 q12.3 q21.1 q23.3 q31.1 q32.2 q33.3 q36.3
2
p25.1 p22.1 p21.1 q11.2 q14.3 q16.3 q22.31 q23.3
-- Access genomic variations beyond SNPs
6
-- Reveal genomic regions inaccessible to
amplification-based sequence methods
contigs > 5Mb
-- Annotate complete gene models with
Contigs <= 5Mb
promoter regions
-- Phase haplotypes with diploid assembly
Mega-base sized contigs were used to piece together nearly
complete chromosome assemblies in the human reference
genome1.
-- Capture epigenetic information
Human (3.2 Gb)
N50: 28 Mb
CONTIG N50 ASSEMBLY STATISTICS OF
GENOMES ASSEMBLED USING ONLY
PACBIO® DATA1-10
Tetraploid Cotton (2.5 Gb)
N50: 219 kb
Bacteria
Finished Genomes
Arabidopsis(120 Mb)
N50: 11 Mb
Panacoid grass (250 Mb)
N50: 2.4 Mb
Sea Bass (700 Mb)
N50: 1 Mb
Goat (2.85 Gb)
N50: 2.5 Mb
Spinach (1 Gb)
N50: 920 kb
Yeast (12 Mb)
Resolve chromosomes
Drosophila (170 Mb)
N50: 21 Mb
Rice (370 Mb)
N50: 4 Mb
p a c b . c o m /d e n o vo
FROM DNA TO COMPREHENSIVE GENOME ASSEMBLIES
Long-Insert Library Preparation
-- Prepare long-insert SMRTbell™ libraries
-- Enrich for longest inserts with size selection options
-- Use recommended gDNA library inputs
-- >5 µg for large genome project
-- Low-input options (100 ng) for microbial and BAC sequencing
-- Support available for library automation
PacBio RS II System
Sequel System
SMRT Sequencing with PacBio Systems
-- Take advantage of the Sequel™ System to reduce project costs
and generate 7X more reads compared with the PacBio RS II
-- Achieve ~10 kb average read lengths, with some reads as long
as 60 kb
-- Scale throughput based on project needs:
-- 8-12X coverage per genome for structural variation surveys
-- 25X coverage per genome for hybrid assembly
-- 50X coverage per genome for PacBio-only de novo assembly
-- Simultaneously capture epigenetic information
Data analysis with SMRT Analysis or PacBio DevNet
-- Create exceptional de novo assemblies with megabase-size
contig N50s and consensus accuracies > 99.999%
-- Generate diploid assemblies with haplotype information for
50-100 large genomes per year
-- Utilize community-developed tools, like Parliament11, to survey
structural variation in larger sample cohorts
KEY REFERENCES
1. http://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP044331
2. Koren, S. and Phillippy, A. M. (2015) One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly. Current
Opinion in Microbiology. 23, 110-120.
3. Kim, K. et al. (2014) Long-read, whole-genome shotgun sequence data for five model organisms. Scientific Data, 1.
4. Berlin, K. et al. (2015) Assembling large genomes with single-molecule sequencing and locality sensitive hashing. Nature Biotechnology. 33(6), 623-630.
5. VanBuren, R. (2015, January) De novo assembly of a complex panicoid grass genome using ultra-long PacBio reads with P6C4 chemistry. Presented at
Plant and Animal Genome XXIII Conference. San Diego, CA.
6. Schatz, M. (2015, January) The resurgence of reference quality genome sequence. Presented at Plant and Animal Genome XXIII Conference. San Diego,
CA.
7. Vij, S. (2014, May) De novo assembly of a medium sized eukaryotic genome using Pacific Biosciences Single Molecule Real Time Sequencing technology.
Presented at Plant and Animal Genome Asia 2014. Singapore.
8. Ashrafi, H. (2015, January). Using spinach to compare technologies for whole genome assemblies. Poster presented at Plant and Animal Genome XXIII
Conference. San Diego, CA.
9. van Eijk, M. (2015, January) Genome assembly and Iso-Seq transcriptome sequencing of tetraploid cotton. Presented at Plant and Animal Genome XXIII
Conference. San Diego, CA.
10.Bickhart, D. (2015, January) The Use of PacBio and Hi-C data in de novo assembly of the goat genome. Presented at Plant and Animal Genome XXIII
Conference. San Diego, CA.
11.English, A. et al. (2015) Assessing structural variation in a personal genome — towards a human reference diploid genome. BMC Genomics. 16, 286.
12.Chin, C. S. et al. (2013) Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nature Methods. 10(6), 563-569.
13.Chaisson, M. J. P. et al. (2014) Resolving the complexity of the human genome using single molecule sequencing. Nature. 517(7536), 608-611.
14.Pendleton, M. et al. (2015) Assembly and diploid architecture of an individual human genome via single-molecule technologies. Nature Methods.
12(8), 780-786.
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