Biome | Q&A with Rich Roberts on single-molecule sequencing technology

The exciting part about single molecule sequencing for me was the ability to sequence low abundance transcripts or have phased haplotypes for human sequencing. Having a high error rate nullifies any advantage in these areas. But I guess it's a tool in the end and how you use it to get meaning results.


"As the previously rapid climb in cost efficiency brought about by next-generation sequencing plateaus, the failure of single-molecule sequencing to deliver might leave some genomics aficionados despondent about the prospects for their field. But a recentCorrespondence article in Genome Biology saw Nobel laureate Richard Roberts, together with Cold Spring Harbor’s Mike Schatz and Mauricio Carneiro of the Broad Institute, argue that the latest iteration of Pacific Biosciences’ SMRT platform is a powerful tool, whose value should be reassessed by a skeptical community.

In this Q&A, Roberts tells us why he thinks there’s a need for re-evaluation, and what sparked his interest in genomics in the first place."

http://www.biomedcentral.com/biome/rich-roberts-discusses-single-molecule-sequencing-technology/

Correspondence

The advantages of SMRT sequencing

Roberts RJ, Carneiro MO and Schatz MC

Genome Biology 2013, 14:405

Go to article >>

Michael Schatz:Assembling Crop Genomes With SMS

PDF of the presentation on Feb 22, 2013 AGBT, Marco Island, FL

http://schatzlab.cshl.edu/presentations/2013-02-20.AGBT.Assembling%20Crop%20Genomes.pdf

if you need an intro

"In a talk during the evening session, Mike Schatz, an assistant professor at Cold Spring Harbor Laboratory, spoke about “Assembling Crop Genomes with Single Molecule Sequencing.” Crops are important to sequence — 15 crops represent 90% of the world’s food, Schatz said — but are notoriously difficult to study because of their large genome size, high repeat content, and higher ploidy. Along with Sergey Koren and Adam Phillippy, he has built a pipeline to create hybrid genome assemblies using PacBio long reads combined with shorter-read sequence — either CCS reads from PacBio or data from another sequencing platform. In an example he offered of a rice strain, an attempted genome assembly using just Illumina reads yielded an N50 contig of 16Kb, but adding PacBio long reads to that boosted the N50 contig to 25Kb. Ultimately, Schatz said, he expects that as PacBio's readlength improves, this kind of approach could routinely generate megabase-size contigs or even pull plant chromosomes into single contigs.

For more information on Mike Schatz’s work using SMRT Sequencing, check out this case studydescribing an automated pipeline for genome finishing with PacBio long reads."

source: http://blog.pacificbiosciences.com/2013/02/notes-from-agbt-long-read-sequence-data.html

He includes a snippet of code to answer this question from twitter

'What's the longest single contig from a de Bruijn assembler without PE or a jumping library?'

$ perl -e 'print ">random\n"; @D=split //,"ACGT"; \for (1...100000000){print $D[int(rand(4))];} \print "\n"’ | fold > random.fa$ wgsim –r 0 -e 0 -N 50000000 -1 100 -2 1 \random.fa random.reads.fq /dev/null$ SOAPdenovo-63mer all –s random.cfg -K 63 -o random.63$ getlengths random.63.contig           1 99999990

OICR's Genomics Pathway-Sequencing Project Highlights Genomic Data's Steady March Into Clinics

OICR's Genomics Pathway-Sequencing Project Highlights Genomic Data's Steady March Into Clinics

The Ontario Institute for Cancer Research has kicked off a project that promises to address some key informatics challenges related to clinical sequencing.

Lincoln Stein, platform leader of informatics and biocomputing at the OICR, described the project in a presentation at the fourth Summit on Translational Bioinformatics in San Francisco this week.

Stein said that the project, conducted in partnership with Canada's Princess Margaret Hospital and dubbed the Genomics Pathway Sequencing project, or GPS, will sequence genes in normal and tumor samples excised from patients.

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“The biggest challenge,” Stein said, “is trying to keep the amount of information in this report to a minimum without keeping potentially important information back [from physicians].”

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For each individual, OICR researchers will sequence about 1,000 cancer-related genes in control and tumor samples and attempt to identify mutations that are of immediate relevance to the patient’s cancer care, or that are targeted by drugs that are currently being tested in trials.

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The genes in the study — which include the usual suspects such as KRAS, p53, and B-Raf — were selected based on suggestions from oncologists in the Toronto area as well as data stored in knowledgebases at the Memorial Sloan Kettering Cancer Center, the Wellcome Trust Sanger Institute, and the National Cancer Institute.

To sequence patient samples, OICR will use Pacific Biosystems' single-molecule sequencing as a first step to discover novel mutations. It will then sequence known mutations on Sequenom's MassArray platform. The sequence variants will be confirmed using a Sanger sequencer housed at the clinical sequencing lab at Princess Margaret hospital.

Stein explained that the OICR selected the PacBio system because “it allows us to do very long reads at high coverage … currently 1,000-base-pair reads in a circular consensus, which gives high accuracy for targeted genes.”

In addition, he said, the platform has a rapid turnaround time, taking only “15 minutes per run to completely analyze … a single typical gene.”

Kevin: fascinating! Would their's become the de facto standard for clinical sequencing?

Pacific Biosciences Team Identifies Asian Origin for Haitian Cholera Bug

Rising importance of NGS for emerging infectious diseases!
excerpted from Bio-ITworld URL above

December 9, 2010 | In a dramatic piece of ultra-quick genetic detective work, next-generation sequencing company Pacific Biosciences has decoded the sequence of the strain of bacteria responsible for the deadly cholera outbreak in Haiti. The findings, which confirm the putative Asian origin for the devastating disease, are published online in the New England Journal of Medicine today. 
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Each of the five strains took about one day to sequence to about 60X coverage. “They did an outstanding job in the analysis,” says Waldor. “Most of the credit for this project goes to Eric and his team.”  
“The rapidity and depth of the sequence using this 3^rd-generation sequencing technology has enormous potential to transform how we can analyze outbreaks of infectious disease and even the prediction of future outbreaks because of the power of their technology.” 
According to PacBio, the five cholera genomes were sequenced on November 12 to 12-15X coverage in less than two hours. Further runs bumped up the coverage to 60X over the course of the day. Over the next three days, the sequence data were subjected to in-depth analysis, including genome assembly, annotation, and sequence comparisons, including comparisons to nearly two dozen published cholera genomes.  
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From PacBio’s perspective, Schadt says that “real-time monitoring” of pathogens opens the door to using his firm’s technology as “a routine surveillance method, for public health protection in addition to pandemic prevention and response.”