Download HIV Forum PacBio Analysis - Forum for Collaborative HIV Research

HIV Forum PacBio Analysis
12 March 2015
FIND MEANING IN COMPLEXITY
PACIFIC BIOSCIENCES® CONFIDENTIAL
© Copyright 2015 by Pacific Biosciences of California, Inc. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Single Molecule, Real-Time (SMRT®) DNA Sequencing
SMRT® Cell
PacBio® RS II
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Zero-Mode
Waveguide
Phospholinked
Nucleotide
Trace
PacBio Sample Preparation and Enhancing Accuracy Using CCS
SMRTbell™ template preparation:
Key Advantages:
• Structurally linear
• Topologically circular
• Structural homogeneity of
templates
• Provides sequences of both
forward and reverse strands in the
same trace
Reads of Insert (ROI) / Circular Consensus Sequencing (CCS)
Generates multiple passes on
each molecule sequenced
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Result: Highly accurate intra-molecular
circular consensus sequence
Examined Datasets
• Examined Datasets:
•
•
Monogram. 28 samples. ~2kb long region
UNC. 4 samples. ~2.8kb long region
• One PacBio Chip Per Sample
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P6/C4 v2 conditions
4h movies
Modified calculator settings (3:1 pol:template ratio)
Pol Reads/Subreads filtered on ≥ 75% predicted accuracy, ≥ 50bp length
CCS filtered on ≥ 6 passes and ≥ 90% predicted accuracy
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Monogram Read Statistics
• Monogram Raw Reads
Number
Reads
Mean
Length
N50
Length
Number
CCS / ROI
MGM
Mean
62140
16910
31060
25360
MGM
Median
60980
17050
31410
28710
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UNC Read Statistics
• UNC Raw Reads
Number
Reads
Mean
Length
N50
Length
Number
CCS / ROI
UNC
Mean
62090
12890
26830
14300
UNC
Median
61440
12710
26860
14220
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Mapped Read Statistics
• Map ROI reads to HXB2 genome for rough mapping statistics
• Monogram:
• 99.9% map (no contamination)
• 1848 base target
• 91% of hits are full length within 2% (truncated PCR products)
• 24,340 mean coverage uniform
• UNC:
• 99.9% map (no contamination)
• 2891 target length
• 80% of hits are full length within 2% (truncated PCR products)
• 13,010 mean coverage uniform
• At 10k coverage and 1% minor, 95% confident minor seen 84 or more times
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De-novo Analysis of UNC Mixtures
• Design: 4 mixes, 8 subspecies at different frequencies, each subspecies two
AA variants
• Use de-novo LAA analysis that yields set of unique sequences estimated to
be in the sample.
o
o
Originally designed for multiple HLA diploid gene sequencing.
Relies on reads covering entire genes so variants are phased by being from a
single molecule from a continuous read
• Take LAA estimates, directly translate to amino acids, place next to reference
amino acid sequence, and call amino acid variants (no alignment)
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De-novo Analysis of UNC Mixtures
• Results with standard parameters.
• Every run calls three sequences of length 2890 bases.
• For 11/12, top three most abundant subspecies called exactly correct. For one
sequence, missed half of one variant.
• Used default settings designed to minimize false positives in general; tuning likely to
yield more sensitive results.
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30
46
54
63
71
82
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L...................D...............M.G.....I........L.......A..........V.I...N.L
GADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIG...
GADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADNTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKVIGTVLVGPTPVNIIGRNLLTQIG...
GADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKLIGGIGGFIKVRQYDQILIEICGHKVIGTVLVGPTPVNIIGRNLLTQIG... 962 AA
GADRQGTVSFSFPQITLWQRPIVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFVKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIG...
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Conclusions and Future Work
• Good coverage of PacBio on HIV Forum Samples
• Long PacBio span regions in their entirety.
• De-novo analysis on mixtures of subspecies
• Works in progress:
• Parameter tuning for more sensitive de novo analysis
• Standard variant positions and haplotype estimation.
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