For Research Use Only. Not for use in diagnostic procedures. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell and Iso-Seq are trademarks of Pacific Biosciences of California, Inc. All other trademarks are the property of their respective owners. © 2014 Pacific Biosciences of California, Inc. All rights reserved. SMRT ® Sequencing and Assembly of the Human Microbiome Project Mock Community Sample – A Feasibility Project Meredith Ashby, Brett Bowman, Cheryl Heiner, Jason Chin Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025 Closed Bacterial Chromosomes Assembly Summary Base Modification Signatures • PacBio data of the HMP Mock Community B assembled with Falcon into 458 contigs; Illumina data assembled with SOAP 1 into ~63,000 contigs. • 99.5% of the reference sequences are contained within just 35 PacBio contigs, including 12 closed bacterial chromosomes. • Examination of the base modification signatures of the contigs revealed 15 of the 19 species for which there was sufficient coverage had unique signatures. • PacBio’s long read lengths, unbiased coverage, high consensus accuracies and ability to detect base modification events are beneficial for improving metagenomics assemblies, allowing for improved functional annotations in metagenome studies. The sample was made into a SMRTbell ™ library with a mean insert size of approximately 12 kb. Fragments <7 kb were removed with BluePippin™ size selection, following standard PacBio ® protocols. The sample was sequenced with a combination of P4-C2 and P5-C3 chemistries. Subread pre-assembly resulted in 1.8 GB of highly accurate reads with a median readlength of 7,033 bp. Introduction Sample Prep While the utility of Single Molecule, Real-Time (SMRT) Sequencing for de novo assembly and finishing of bacterial isolates is well established, this technology has not yet been widely applied to shotgun sequencing of microbial communities. In order to demonstrate the feasibility of this approach, we sequenced genomic DNA from the Microbial Mock Community B of the Human Microbiome Project Assembly Details Conclusions Escherichia coli K-12 MG1655 Helicobacter pylori 26695 Deinococcus radiodurans R1 Listeria monocytogenes EGD-e Neisseria meningitidis MC58 Bacillus cereus ATCC 10987 Streptococcus agalactiae 2603V/R Streptococcus mutans UA159 Propionibacterium acnes KPA171202 Rhodobacter sphaeroides 2.4.1 Lactobacillus gasseri ATCC 33323 Enterococcus faecalis OG1RF Reads: 1.4 M Filtered Data: 6.6 GB Mean: 4,846 bp N50: 6,811 Reads: 5.7 M Filtered Data: 13.1 GB Mean: 2,294 bp N50: 3,526 The PacBio data was assembled with a combination of HGAP and Falcon. The selected PacBio results below are compared to a published SOAP assembly using Illumina ® data. 1 Contigs are separated by horizontal lines in the bar plot. Bacteria Reference PacBio Contigs Illumina Contigs 1 Reference Length PacBio Asm. Length Illumina Asm. Length 1 Acinetobacter baumannii ATCC 17978 NC_009085.1 2 98 3,976,747 4,062,673 3,938,117 Actinomyces odontolyticus ATCC 17982 NZ_DS264586.1 2 787 2,391,230 2,396,710 1,594,838 Bacillus cereus ATCC 10987* NC_003909.8 1 3 5,224,283 5,192,114 3,978 Bacteroides vulgatus ATCC 10987 NC_009614.1 7 243 5,163,189 5,128,316 5,025,345 Clostridium beijerinckii NCIMB 8052 NC_009617.1 3 1,605 6,000,632 5,985,675 2,493,854 Deinococcus radiodurans R1 NC_001263.1 2 343 2,648,638 2,654,395 2,622,689 NC_001264.1 1 47 412,348 423,234 408,658 Enterococcus faecalis OG1RF NC_017316.1 1 883 2,739,625 2,750,252 1,403,967 Escherichia coli K-12 MG1655* NC_000913.3 1 176 4,641,652 4,664,208 219,711 Heliobacter pylori NC_000915.1 1 81 1,667,867 1,678,033 1,609,609 Lactobacillus gasseri ATCC 33323* NC_008530.1 1 - 1,894,360 1,850,783 NA Listeria monocytogenes EGD-e NC_003210.1 1 869 2,944,528 2,956,639 2,652,834 Neisseria meningitidis MC58 NC_003112.2 1 685 2,272,360 2,266,612 1,701,827 Propionibacterium acnes KPA171202 NC_006085.1 1 192 2,560,265 2,571,155 2,534,743 Pseudomonas aeruginosa PA01* NC_002516.2 1 3 6,264,404 6,321,442 3,802 Rhodobacter sphaeroides 2.4.1* NC_007493.2 3 373 3,188,524 3,188,332 557,568 NC_007494.2 1 96 943,018 931,082 153,761 Staphylococcus aureus USA300_TCH1516 NC_010079.1 2 181 2,872,915 2,895,692 2,844,516 Staphylococcus epidermidis ATC 12228 NC_004461.1 2 109 2,499,279 2,513,932 2,419,062 Streptococcus agalactiae 2603V/R* NC_004116.1 1 - 2,160,267 2,166,843 NA Streptococcus mutans UA159 NC_004350.2 1 188 2,032,925 2,058,865 1,974,377 Streptococcus pneumoniae TIGR4* NC_003028.3 22 209 2,160,842 NA 2,019,766 *Sample prep variability or sequencing depth resulted in very low coverage from these species in either the Illumina or PacBio studies Bacteria Mean Coverage Base Modification Signature Acinetobacter baumannii ATCC 17978 56.30 None Actinomyces odontolyticus ATCC 17982 85.79 R A GCNNNNNNCGT / A CGNNNNNNGCTY G A YNNNNNNTAYG/ CRT A NNNNNNRTC CTCG A G Bacillus cereus ATCC 10987 37.23 CC A NNNNNNNCTTA / TA A GNNNNNNNTGG CGA A G Bacteroides vulgatus ATCC 10987 85.60 CYY A NNNNNNNCTTG / CA A GNNNNNNNTRRG C A CNNNNNRTG / C A YNNNNNGTG Clostridium beijerinckii NCIMB 8052 42.26 CNT A YNNNNNNCTTC / GA A GNNNNNNRTANG Deinococcus radiodurans R1 92.56 C C GCGG Enterococcus faecalis OG1RF 76.15 None Escherichia coli K-12 MG1655 66.69 GC A CNNNNNNGTT / A A CNNNNNNGTGC G A TC Heliobacter pylori 408.06 G A GG GAAG A ATTA A T TCG A C A TG G A TC DGA A GG GC A G G A NTC G C GC TCTT C AC A NNNNNNNNTAG / CT A NNNNNNNNTGT Lactobacillus gasseri ATCC 33323 113.74 T A CNNNNNCTC / G A GNNNNNGTA Listeria monocytogenes EGD-e 124.32 GG C C Neisseria meningitidis MC58 102.29 G A CGC C CWCC? Propionibacterium acnes KPA171202 111.91 AGC A GY Pseudomonas aeruginosa PA01 91.56 G A TCNNNNNNGTC / G A CNNNNNNGATC Rhodobacter sphaeroides 2.4.1 47.87 G A NTC Staphylococcus aureus USA300_TCH1516 105.02 A GGNNNNNGAT / A TCNNNNNCCT AC A NNNNNNRTGG / CC A YNNNNNNTGT Staphylococcus epidermidis ATC 12228 91.33 None Streptococcus agalactiae 2603V/R 54.21 None Streptococcus mutans UA159 121.39 RG A NNNNNNNTCG / CG A NNNNNNNTCY CTGR A G / CTYC A G G A TC CTGC A G Streptococcus pneumoniae TIGR4* - - References 1. Treangen, T.J., Koren, S., Sommer, D.D., Liu, B., Astrovskaya, B.O., Darling, A.E., Phillipy, A.M., Pop, M. (2013) MetAMOS: A modular and open source metagenomic assembly and analysis pipeline. Genome Biology 14:R2. PacBio provides the unique opportunity to study base modification in genomic DNA while sequencing. The results below were generated with no additional sample prep. In the HMP sample, 19 species had enough coverage to examine base modification, with 15 species showing unique signatures.
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For Research Use Only. Not for use in diagnostic procedures. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell and Iso-Seq are trademarks of Pacific Biosciences of California, Inc. All other trademarks are the property of their respective owners. © 2014 Pacific Biosciences of California, Inc. All rights reserved.
SMRT® Sequencing and Assembly of the Human Microbiome Project Mock Community Sample – A Feasibility ProjectMeredith Ashby, Brett Bowman, Cheryl Heiner, Jason ChinPacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025
Closed Bacterial Chromosomes Assembly Summary Base Modification Signatures
• PacBio data of the HMP Mock Community B assembled with Falcon into 458 contigs; Illumina data assembled with SOAP1 into ~63,000 contigs.
• 99.5% of the reference sequences are contained within just 35 PacBio contigs, including 12 closed bacterial chromosomes.
• Examination of the base modification signatures of the contigs revealed 15 of the 19 species for which there was sufficient coverage had unique signatures.
• PacBio’s long read lengths, unbiased coverage, high consensus accuracies and ability to detect base modification events are beneficial for improving metagenomics assemblies, allowing for improved functional annotations in metagenome studies.
The sample was made into a SMRTbell™ library with a mean insert size of approximately 12 kb. Fragments <7 kb were removed with BluePippin™ size selection, following standard PacBio® protocols. The sample was sequenced with a combination of P4-C2 and P5-C3 chemistries. Subread pre-assembly resulted in 1.8 GB of highly accurate reads with a median readlength of 7,033 bp.
Introduction
Sample Prep
While the utility of Single Molecule, Real-Time (SMRT) Sequencing for de novo assembly and finishing of bacterial isolates is well established, this technology has not yet been widely applied to shotgun sequencing of microbial communities. In order to demonstrate the feasibility of this approach, we sequenced genomic DNA from the Microbial Mock Community B of the Human Microbiome Project
Assembly Details
Conclusions
Escherichia coli K-12 MG1655 Helicobacter pylori 26695 Deinococcus radiodurans R1
Listeria monocytogenes EGD-eNeisseria meningitidis MC58 Bacillus cereus ATCC 10987
Streptococcus agalactiae 2603V/R
Streptococcus mutans UA159 Propionibacterium acnes KPA171202
Rhodobacter sphaeroides 2.4.1 Lactobacillus gasseri ATCC 33323
Enterococcus faecalis OG1RF
Reads: 1.4 MFiltered Data: 6.6 GBMean: 4,846 bpN50: 6,811
Reads: 5.7 MFiltered Data: 13.1 GBMean: 2,294 bpN50: 3,526
The PacBio data was assembled with a combination of HGAP and Falcon. The selected PacBio results below are compared to a published SOAP assembly using Illumina®
data.1 Contigs are separated by horizontal lines in the bar plot.
Bacteria Reference PacBio Contigs
Illumina Contigs 1
Reference Length
PacBio Asm. Length
Illumina Asm. Length 1
Acinetobacter baumannii ATCC 17978 NC_009085.1 2 98 3,976,747 4,062,673 3,938,117
Actinomyces odontolyticus ATCC 17982 NZ_DS264586.1 2 787 2,391,230 2,396,710 1,594,838
Bacillus cereus ATCC 10987* NC_003909.8 1 3 5,224,283 5,192,114 3,978
Bacteroides vulgatus ATCC 10987 NC_009614.1 7 243 5,163,189 5,128,316 5,025,345
Clostridium beijerinckii NCIMB 8052 NC_009617.1 3 1,605 6,000,632 5,985,675 2,493,854
Deinococcus radiodurans R1NC_001263.1 2 343 2,648,638 2,654,395 2,622,689
NC_001264.1 1 47 412,348 423,234 408,658
Enterococcus faecalis OG1RF NC_017316.1 1 883 2,739,625 2,750,252 1,403,967
Escherichia coli K-12 MG1655* NC_000913.3 1 176 4,641,652 4,664,208 219,711
Heliobacter pylori NC_000915.1 1 81 1,667,867 1,678,033 1,609,609
Lactobacillus gasseri ATCC 33323* NC_008530.1 1 - 1,894,360 1,850,783 NA
Listeria monocytogenes EGD-e NC_003210.1 1 869 2,944,528 2,956,639 2,652,834
Neisseria meningitidis MC58 NC_003112.2 1 685 2,272,360 2,266,612 1,701,827
Propionibacterium acnes KPA171202 NC_006085.1 1 192 2,560,265 2,571,155 2,534,743
Pseudomonas aeruginosa PA01* NC_002516.2 1 3 6,264,404 6,321,442 3,802
Rhodobacter sphaeroides 2.4.1*NC_007493.2 3 373 3,188,524 3,188,332 557,568
NC_007494.2 1 96 943,018 931,082 153,761Staphylococcus aureus USA300_TCH1516 NC_010079.1 2 181 2,872,915 2,895,692 2,844,516
Staphylococcus epidermidis ATC 12228 NC_004461.1 2 109 2,499,279 2,513,932 2,419,062
Streptococcus agalactiae 2603V/R* NC_004116.1 1 - 2,160,267 2,166,843 NA
Streptococcus mutans UA159 NC_004350.2 1 188 2,032,925 2,058,865 1,974,377
Streptococcus pneumoniae TIGR4* NC_003028.3 22 209 2,160,842 NA 2,019,766*Sample prep variability or sequencing depth resulted in very low coverage from these species in either the Illumina or PacBio studies
Bacteria Mean Coverage Base Modification Signature
Acinetobacter baumannii ATCC 17978 56.30 None
Actinomyces odontolyticus ATCC 17982 85.79RAGCNNNNNNCGT / ACGNNNNNNGCTYGAYNNNNNNTAYG/ CRTANNNNNNRTCCTCGAG
Bacillus cereus ATCC 10987 37.23CCANNNNNNNCTTA / TAAGNNNNNNNTGGCGAAG
Bacteroides vulgatus ATCC 10987 85.60CYYANNNNNNNCTTG / CAAGNNNNNNNTRRGCACNNNNNRTG / CAYNNNNNGTG
Clostridium beijerinckii NCIMB 8052 42.26 CNTAYNNNNNNCTTC / GAAGNNNNNNRTANG
Deinococcus radiodurans R1 92.56 CCGCGG
Enterococcus faecalis OG1RF 76.15 None
Escherichia coli K-12 MG1655 66.69GCACNNNNNNGTT / AACNNNNNNGTGCGATC
Heliobacter pylori 408.06
GAGG GAAGA ATTAAT TCGACATG GATC DGAAGGGCAG GANTC GCGC TCTTCACANNNNNNNNTAG / CTANNNNNNNNTGT
Lactobacillus gasseri ATCC 33323 113.74 TACNNNNNCTC / GAGNNNNNGTAListeria monocytogenes EGD-e 124.32 GGCCNeisseria meningitidis MC58 102.29 GACGC CCWCC?Propionibacterium acnes KPA171202 111.91 AGCAGYPseudomonas aeruginosa PA01 91.56 GATCNNNNNNGTC / GACNNNNNNGATC
Rhodobacter sphaeroides 2.4.1 47.87 GANTC
Staphylococcus aureus USA300_TCH1516 105.02 AGGNNNNNGAT / ATCNNNNNCCTACANNNNNNRTGG / CCAYNNNNNNTGT
Staphylococcus epidermidis ATC 12228 91.33 NoneStreptococcus agalactiae 2603V/R 54.21 None
Streptococcus mutans UA159 121.39RGANNNNNNNTCG / CGANNNNNNNTCYCTGRAG / CTYCAG GATC CTGCAG
Streptococcus pneumoniae TIGR4* - -
References1. Treangen, T.J., Koren, S., Sommer, D.D., Liu, B., Astrovskaya, B.O., Darling, A.E., Phillipy, A.M.,
Pop, M. (2013) MetAMOS: A modular and open source metagenomic assembly and analysis pipeline. Genome Biology 14:R2.
PacBio provides the unique opportunity to study base modification in genomic DNA while sequencing. The results below were generated with no additional sample prep. In the HMP sample, 19 species had enough coverage to examine base modification, with 15 species showing unique signatures.
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