1 nicholas mancuso department of computer science georgia state university joint work with bassam...

Nicholas MancusoDepartment of Computer Science

Georgia State University

Joint work with Bassam Tork, GSUPavel Skums, CDCIon Mӑndoiu, UConn Alex Zelikovsky, GSU

Viral Quasispecies Reconstruction from Amplicon 454 Pyrosequencing

CAME 2011, Atlanta Georgia

Viral Quasispecies and NGS• RNA Viruses

—HIV, HCV, SARS, Influenza—Higher (than DNA) mutation rates — quasispecies

—set of closely related variants rather than a single species

• Knowing quasispecies can help—Interferon HCV therapy effectiveness (Skums et al 2011)

• NGS allows to find individual quasispecies sequences—454 Life Sciences : 400-600 Mb with reads 300-800 bp long

• Sequencing is challenging—multiple quasispecies —qsps sequences are very similar

—different qsps may be indistinguishable for > 1kb (longer than reads)

Outline

• Shotgun vs Amplicon Sequencing• Viral Quasispecies Reconstruction Problem• Challenges and Approaches• Data Structure for Reads: Read Graph• Novel Methods for Solving QSR Problem• Observed vs True Read Frequencies• True Frequency Reconstruction• Simulations and Results

Shotgun versus Amplicon Sequencing

• Shotgun reads—starting positions

distributed uniformly

• Amplicon—each read has

predefined start/endcovering fixed overlappingwindows

Viral Quasispecies Spectrum Reconstruction Problem

• Given—collection of amplicon reads from a quasispecies

population with unknown variants and distribution

• Find—viral quasispecies sequences and their frequencies

Amplicon Sequencing Challenges• Collapse of quasispecies in amplicon

—distinct quasispecies may be indistinguishable in window• Collapse of quasispecies in overlap

—match reads from consecutive windows coming from the same qsp

• First approachProsperi et al (2011)—Guide Distribution

—choose a column—go right/left matching the

the closest in order neighbor

220 200 140 160 150200 140 130 150 14070 130 120 140 13010 20 110 130 1200 10 100 20 60

Approaches to QSP Reconstruction • Shotgun approaches

—estimates probability of consecutive reads coming from the same qsp (ViSpA, Astrovskaya et al 2011)

—parsimony (minimum number of distinct sequences covering all reads) (ShoRAH, Zagordi et al 2010)

• Why not use shotgun approaches for amplicons?—estimating probability in ViSpA relies on uniform distribution of reads—amplicon reads have fixed beginnings and ends

• Optimization approach—most parsimonious solution

— minimize number of distinct sequences covering all reads — too coarse: many different optimal solutions

—minimum information entropy (Shannon, 1948)— takes in account also frequency— fractional relaxation of pure parsimony

Min Entropy vs Parsimony• Parsimony and Min Entropy selects AC and BD if a = c, and b = d

8 CAME 2011, Atlanta Georgia

Data Structure for Reads: Read GraphK amplicons → K-staged read graph

—vertices → distinct reads—edges → reads with consistent overlap—vertices, edges have a count function

Read Graph• May transform graph into a 'forked' graph

—overlap is represented by fork vertex

Fork Resolving Problem• Minimum Entropy is NP-hard

—can solve it optimally for each small fork separately (future work)

• Greedy heuristic— ≤ a+b-1 are sufficient when resolving fork with a distinct reads on the

left and b on the right— that can be done greedily matching largest (greedy heuristic)— this does not guarantee minimum number of distinct qsps

• Better way = globally match the most frequent reads (max bandwidth)— find s-t path maximizing minimum read count— subtract the minimum count from each read in the path

— exhausts at least one read in the path

Greedy Method

Maximum Bandwidth Method

Observed vs Ideal Read Frequencies• Ideal frequency

—consistent frequency across forks

• Observed frequency (count)—inconsistent frequency across forks

• All methods perform better under ideal frequencies

Fork Balancing Problem

• Given—set of reads and respective frequencies

• Find—minimal frequency offsets balancing all forks

Simplest approach is to scale frequencies from left to right

Least Squares Approach

• Quadratic Program for read offsets• q – fork, oi – observed frequency, xi – frequency offset

Flowchart

Data Sets and Metrics• Simulated error-free HCV (1734 long fragment)– quasispecies from uniform, geometric, and skewed distribution– shift → delta of starting position

• Sensitivity– percentage of correctly assembled true quasispecies

• PPV– percentage of true quasispecies among all assembled

• Jensen-Shannon Divergence

Sensitivity Results

PPV Results

Divergence Results

ViSpA Comparison

Conclusion

• Two novel methods for solving QSR problem—Outperform Prosperi et al. on average—Outperform ViSpA approach on average

• Maximum Bandwidth approach worked best

• Future work: exact local solution for minimum entropy

Thanks

1 nicholas mancuso department of computer science georgia state university joint work with bassam...

atlanta georgia slide

amplicon distinct quasispecies

reads shorah

viral quasispecies sequences

quasispecies population

graph vertices distinct

amplicon sequencing

amplicon sequencing

Documents

catálogo tork

eng. bassam mansour

Каталог tork

page 16 july 23, 2008 louis michael mancuso sr. ·...

mancuso - cap iv

tes 354 elektrikli tork anahtari tork tablosu 1

jeannette mancuso

bassam profile

susan h. mancuso, crfe susan mancuso consulting ·...

Χαρτοπετσέτες tork

mc bassam update

get ready for business with the right tork products · tork...

by bassam hussein

tork katalógus

tork extra mild liquid soap tork жидкое...

bassam tork georgia state university atlanta, ga 30303, usa

amplicon-based quasipecies assembly using next generation...

Каталог продукции tork

catalogue tork

tork performance