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  • Single’molecule,’Real/Time’(SMRT)’DNA sequencing’– Sequel ......Sample3 BacteriaGram /’’ ’GC%:’52.1’ Sample4 Fungi’’ Genomesize 4.086’Mb’ 4.8’Mb’
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Single molecule, RealTime (SMRT) DNA sequencing – Sequel PacBio® Biomics Pole h"p://citech.pasteur.fr/team/biomics/ Applica>on Whole Genome sequencing Future Microbial Mul>plexing sequencing 1 SMRT Cell / 16 microbes (<2 Mb) 1 SMRT Cell / 12 microbes (<5 Mb) 1 Million wells called Zeromode wave guides (ZMWs) Performance Sequel Throughtput: 45 Gb Average read length: 1018 kb Movie >mes: 30 mins to 10 hours Input : gDNA First results obtained with the Sequel chemistry v2.0 (Febuary 2017) Metrics Sequel Sample 1 Bacteria Gram GC% 67.7 Sample 2 Bacteria Gram GC%: 64.1 Sample 3 Bacteria Gram GC%: 52.1 Sample 4 Fungi Genome size 4.086 Mb 4.8 Mb 5.06 Mb 14.58 Mb Size selec>on library 15 Kb 10 Kb 15 Kb 15 Kb Nbr of bases 2.7 Gb 2.64 Gb 6.72 Gb 4.77 Gb Nbr of Polymerase reads 186,254 317,385 514,545 390,011 Polymerase read length (Mean) 14,512 8,342 13,064 12,234 Polymerase read N50 23,750 13,250 21,250 20,250 Insert length (Mean) 11,832 6,512 10,982 10,262 Metrics Value Number of subread bases mapped 2,333 Gb Number of subreads (mapped) 187,999 Subread length Mean (mapped) 12,410 Subread length N50 (mapped) 16,821 Subread length Max (mapped) 47,623 Mapped concordance (mean) 86.63 Sample 1 Resequencing analysis – SMRT Link v4 1 SMRT Cell per library – 10 hours movie Consensus accuracy: 99.998 Mean base coverage: 545 Library construc>on Quality control of gDNA Nanodrop and Qubit measurements Fragment Analyzer profil (to ~50kb) Pulse Field Gel Electrophoresis

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Page 1: Single’molecule,’Real/Time’(SMRT)’DNA sequencing’– Sequel ......Sample3 BacteriaGram /’’ ’GC%:’52.1’ Sample4 Fungi’’ Genomesize 4.086’Mb’ 4.8’Mb’

Single  molecule,  Real-­‐Time  (SMRT)  DNA  sequencing  –  Sequel  PacBio®    

Biomics Pole h"p://citech.pasteur.fr/team/biomics/  

Applica>on    Whole  Genome  sequencing  

Future  Microbial  Mul>plexing  sequencing  1  SMRT  Cell  /  16  microbes  (<2  Mb)  -­‐  1  SMRT  Cell  /  12  microbes  (<5  Mb)  

1  Million  wells  called  Zero-­‐mode  wave-­‐guides  (ZMWs)  

Performance  Sequel    Throughtput:  4-­‐5  Gb  Average  read  length:  10-­‐18  kb  Movie  >mes:  30  mins  to  10  hours  

Input  :  gDNA  

First  results  obtained  with  the  Sequel  chemistry  v2.0  (Febuary  2017)    

Metrics  Sequel  

Sample  1  Bacteria  Gram-­‐      

GC%  67.7  

Sample  2  Bacteria  Gram-­‐    

 GC%:  64.1  

Sample  3  Bacteria  Gram-­‐      GC%:  52.1  

Sample  4  Fungi    

Genome  size   4.086  Mb   4.8  Mb   5.06  Mb   14.58  Mb  

Size  selec>on  library  

15  Kb   10  Kb   15  Kb   15  Kb  

Nbr  of  bases   2.7  Gb   2.64  Gb   6.72  Gb   4.77  Gb  

Nbr  of  Polymerase  reads  

186,254   317,385   514,545   390,011  

Polymerase  read  length  (Mean)  

14,512   8,342   13,064   12,234  

Polymerase  read  N50   23,750   13,250   21,250   20,250  

Insert  length  (Mean)   11,832   6,512   10,982   10,262  

Metrics    Value  

Number  of  subread  bases  mapped   2,333  Gb  

Number  of  subreads  (mapped)   187,999  

Subread  length  Mean  (mapped)   12,410  

Subread  length  N50  (mapped)   16,821  

Subread  length  Max  (mapped)   47,623  

Mapped  concordance  (mean)   86.63  

Sample  1  Resequencing  analysis  –  SMRT  Link  v4  

1  SMRT  Cell  per  library  –  10  hours  movie  

Consensus  accuracy:  99.998  Mean  base  coverage:  545  

Library  construc>on    

Quality  control  of  gDNA  -­‐  Nanodrop  and  Qubit  

measurements  -­‐  Fragment  Analyzer  profil  (to  

~50kb)  -­‐  Pulse  Field  Gel  Electrophoresis    

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