New Technologies at the Center for Bioinformatics & Functional Genomics at Miami University
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The document outlines the resources and technologies available at the Center for Bioinformatics & Functional Genomics (CBFG) as of August 2017, including training requirements, laboratory facilities, and a comprehensive inventory of instrumentation. It highlights advancements in sequencing technologies such as Sanger sequencing and Next Generation Sequencing (NGS), including applications for microbiomes and metagenomics. Additionally, the document details data analysis protocols and resources for differential gene expression analysis and robotics for laboratory automation.
New Technologies at the Center for Bioinformatics & Functional Genomics at Miami University
- 1. From minIONs to Microbiomes:From minIONs to Microbiomes: New Technologies at the CBFG and How They Can Help Your Research Programme Andor J Kiss Director – Center of Bioinformatics & Functional Genomics August 31st , 2017
- 2. 2 Where, What, and How? ● CBFG is located in the lower floor of Pearson Hall – 086 Pearson Hall ● Three main rooms: ● Main wet laboratory – Swipe Card Entry ● Conference Room (Collaboration) – Key Entry ● Computer Room – Key Entry
- 3. 3 Where, What, and How? Pearson Hall Hallway 086A Conference Room 086B Computer Room / XD Sequencers BiomekFX Main Wet Laboratory Director’s Office
- 4. 4 Where, What, and How? ● Training facility – you do the work, we help… – Need to be trained by us – Not your lab mates ● Only use instrumentation on which you’ve been trained ● Do not prop door open afer hours ● Do not admit other people afer hours ● Only responsible for consumables – No “rental” fees – No surcharges
- 5. 5 CBFG ~ Comprehensive Inventory & Description ● Available on-line via the FTP website ● Also, this presentation will be on-line as well ● Comprehensive list and brief description ● Cost to use is best discussed in person ● If you need instrumentation and we do not have: (a)Consider writing an MRI, or other external grant proposal. (b)If it’s small (< $35,000), Student Tech Fee? (c)Maybe we can “pass the hat” and find the money. ● We will not duplicate instrumentation
- 6. 6 What’s on the FTP Server? ● Data (Sequencing) ● Protocols – CBFG – Industry Standard ● Sofware – Bioinformatics – Instrumentation ● PDF Papers – MIQE qPCR Guideline(s) ● Chinese
- 7. 7 What’s New? Old? ● Sanger Sequencing – ABI3130xl ● 16 channel; ~1000 bases – ABI3730 ● 48 channel; ~700 bases ● Nanodrops – ND1000, ND2000 ● UV/VIS – ND3300 ● Fluorospectrophotometer ● qPCR / PCR – CFX Connect, Rotorgene3000 – MJ Research, VERTI ● Robotics – BiomexFX ● microplate (96/384) ● Liquid Handler ● Open system – Maxwell16 ● Promega – (16 samples max) ● Magnetic Bead ● Pre-defined Kits ● Conference Room – New LED Lights – Dimmable, cool (no heat)
- 8. 8 What’s New? Old? ● Computers – Three Linux machines ● facilitates open-source bioinformatics sofware ● ECC RAM – CLC Genomics Workbench – CLC Main Workbench – Blast2GO PRO – Biogazelle qBasePlus – GeneCodes Sequencer – CANOCO (GUI version of “R”) – PAUP*4b10
- 9. 9 Blast2GO PRO → Gene Ontology Annotation Sofware ● Annotates cDNAs with functionality of the gene ● Sorts by GO ● Common / Unique GO ● Enrichment of GO? – Which GO goes ↑ – Which GO goes ↓
- 10. 10 What’s New? ● Pulse Field Gel Electrophoresis (PFGE) – 500 bp ~ 4.0 Mbp separation range – Excellent for genomics work – Ideal for isolating large fragments – Ideal for RLFP, Southerns ● VWR UV/VIS Spectrophotometer – Peltier/Sipper Unit – Jacketed 1 & 4 position – UV & Glass Flow Cell – Kinetics, Quantitation, Scan, DNA/Protein, Multiwavelength
- 11. 11 What’s New? ● Next Generation Sequencing (NGS) – Illumina MiSeq – ONT minION – Covaris M220 Ultrasonicator – Sage PippinPrep – Bioanalyzer2100 – ND3300 Fluorospectrophotometer – Magnetic Stands
- 12. 12 What is ‘NGS’ and how does it difer from Sanger? ● Sanger sequencing (BigDye, Cycle Sequencing) – One template per reaction – Labour intensive – Expensive – Slow – Long reads (>700 bp) – Very high quality ● “gold standard” of DNA sequencing
- 13. 13 Sanger Sequencing – Radio-labelled 35 S
- 14. 14 Sanger Sequencing – BigDye 3.1 (ABI) By Estevezj - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=23264166
- 15. 15 NGS, or more specifically ‘SBS’: Sequencing-By-Synthesis ● Developed in 1998 – Commercialised by “Solexa” ● Acquired by Illumina 2007 – Greatly improved the original method ● NGS market 4.8 billion (2016) – 12 billion by 2021 ● Annual Growth Rate +16% ● Illumina is ~75% global market
- 16. 16 SBS: Sequencing By Synthesis ● Sample Preparation – DNA Fragments + Oligonucleotides (Complimentary to FLOW CELL) ● Cluster Generation – DNA fragments are hybridised to FLOW CELL – Bridge amplification → Clusters ● Sequencing By Synthesis – Extension of primer based on Watson-Crick pairing – Uses modified bases that emit light → “Illumina” ● Assembly of the SBS Data – Fragments are random; each has a diferent STOP / START – ASSEMBLERS overlap the fragments into large contiguous pieces → CONTIGS
- 17. 17 How Does SBS Work?
- 18. 18 SBS: Key Step → Bridge Amplification & Clusters
- 19. 19 SBS: Multiple Platforms → Scale & Application ● CBFG has a MiSeq (most versatile; cost efective) – $100K instrument; $1100 ~ $1500 per SBS run ● SBS chemistry is vertically integrated – Libraries made for MiSeq can be sequenced on HiSeqs*
- 20. 20 MiSeq Recommended Applications (Supported)
- 21. 21 MiSeq: Microbiomes and/or Metagenomics ● Microbiome – Microbial component of the human gut* – Microbial composition of a tissue or organ system ● Changes with: – Diet – Race – Time – Stress / Disease
- 22. 22 MiSeq: Microbiomes & Metagenomics ● Schloss Group (mothur) – 2x250 bp | almost full over-lap | error correction – Choose OTUs first; compare to reference second – Newer project; highest DATA quality paramount ● Gilbert / Knight / Caporaso (QIIME/emp) – 2x150 bp | ~20-25 bp overlap | little error correction – Choose OTUs by comparison to reference – Older project; consistency of the method
- 23. 23 MiSeq: 16S microbiome & metagenomics http://omegabioservices.com/index.php/16s-reference/
- 24. 24 MiSeq: 16S microbiome & metagenomics http://www.idtdna.com/pages/decoded/decoded-articles/handling-oligos/decoded/2017/07/07/16s-rrna-indexed-primers-amplify-p hylogenic-markers-for-microbiome-sequencing-analysis
- 25. 25 MiSeq: Amplicon Sequencing → Any loci Target Loci
- 26. 26 MiSeq: Amplicon Sequencing → Any loci From: “Overview of tailed amplicon sequencing approach with MiSeq”. Jonathan Bell. Illumina (2011). Reduces the amount of PhiX spike-in required, decreases cluster %PF
- 27. 27 MiSeq: Demultiplexing → “binning reads” ● Sequence data is acquired as FASTQ – FASTA (the actual A, C, T, and G) and a “Q-Score” ● The “barcode” is now called “INDEX” ● INDEX sequences are common to each SAMPLE ● Each SAMPLE is “binned” by INDEX code – Bioinformatically, afer sequence acquisition – computer.
- 28. 28 MiSeq: 16S rRNA + ????? ● 18S rDNA (eukaryotic) ● Small Sub-Unit of rRNA | SSU rDNA V9 gene (1391f/1510r) ● ITS (yeast) ● Intertransgenic Spacer (between the 18S rRNA and 5.8S rRNA genes) ● ITS1 (ITSf1/ITS2) ● Any loci ● <you_provide_target_primer> ● 2b RAD-Seq or “RAD-Seq” approaches ● These are becoming extremely popular for population studies ● Evolution & Ecological Genomics → Do NOT require a reference genome
- 29. 29 MiSeq: RAD-Seq based technologies ● 2b RAD-Seq – Essentially combines RFLP mapping with NGS – Do NOT need a reference genome – Is extremely sensitive ● Good for population genetics ● Identify SNPs – Easy to perform Wang et al. (2012) Nature Methods. doi:10.1038/nmeth.2023 Fig. 1
- 30. 30 MiSeq: 16S rDNA NGS Sequencing (SOP) 23 Page, extremely detailed “How To” complete with 27 8x10 colour glossy pictures with circles and arrows and a paragraph explaining what each one means.
- 31. 31 MiSeq: 16S rDNA Sequencing (Amplicon) ● Schloss Lab – University of Michigan – Dept Pediatrics ● Microbiome focussed – Disease correlations – novel OTUs ● Jack Gilbert – Argonne National Labs ● Rob Knight – UC San Diego ● Guts & Non-Guts https://www.mothur.org/ http://qiime.org/
- 32. 32 MiSeq: Microbiome & Metagenome Analysis ● Open-source – mothur – QIIME ● Closed Source / Paid – CLC Microbial Genomics Module ● Qiagen Microbial Genomics Pro Suite ● CLC Genomics Workbench – Services ● Paid bioinformatics data processing
- 33. 33 MiSeq: Microbiome & Metagenomics Analysis CLC Genomics Workbench – Microbial Genomics Module
- 34. 34 MiSeq: 16S rDNA “Canine Oral Microbiome” Are dog’s mouths cleaner than humans? No, and they have diferent flora. Moraxellaceæ Pasteurellaceæ Burkholderiaceæ
- 35. 35 MiSeq: 16S rDNA Polar Lake Metagenomics Teufel, A. G., Li, W., Kiss, A. J. & Morgan-Kiss, R. M. (2016) Impact of nitrogen and phosphorus on phytoplankton production and bacterial community structure in two stratified Antarctic lakes: a bioassay approach. Polar Biol. doi:10.1007/s00300-016-2025-8
- 36. 36 MiSeq: 16S rDNA Polar Lake Metagenomics ● Relate Lake characteristics to species → those to other related (interdependent) species
- 37. 37 NGS: Diferential Gene Expression Analysis ● Isolate totalRNA ● Clean the totalRNA of rRNA – poly(A)+ selection and/or probe pull-down (Ribo-Zero) ● Convert the mRNA to cDNA ● Add ADAPTORS + INDEX codes for sequencing – Platform dependent ● Amplify the cDNA using PCR ● Acquire the DNA sequence
- 38. 38 NGS: Diferential Gene Expression Analysis ● Isolate totalRNA ● Clean the totalRNA of rRNA – poly(A)+ selection and/or probe pull-down (Ribo-Zero) ● Convert the mRNA to cDNA ● Add ADAPTORS + INDEX codes for sequencing – Platform dependent ● AMPLIFY THE cDNA USING PCR ● Acquire the DNA sequence
- 39. 39 NGS: Diferential Gene Expression Analysis ● How to mitigate PCR bias in NGS experiments? (1) Good library construction practices. (2) Minimise PCR cycles. (3) Increase: Number of ‘biological replicates’ (# of animals) Number of ‘technical replicates’ (# of libraries per animal) • Sometimes this is not possible: • Poor quality / low amounts of totalRNA • Single cell samples
- 40. 40 NGS: Diferential Gene Expression Analysis ● One possible solution is: ● ‘molecular indexing’ ● ‘unique molecular indices’ ● ‘molecular tags’ ● ‘unique tags’ ● ‘fragment tags’ ● Prior to PCR amplification: – Add UMIs to each fragment – Randomly on both ends – Separate from the INDEX codes → per sample
- 41. 41 NGS: DGE Analysis & UMIs Adapted from BIOO Scientific NEXTflex® qRNA-Seq™ Description
- 42. 42 NGS: DGE Analysis & UMIs Adapted from BIOO Scientific NEXTflex® qRNA-Seq™ Description ● Multiple manufacturers – Cellular Research, BIOO (PE), Archer, Swif, NEB, ● Slightly diferent design ● UMIs added prior to PCR – Molecule dependent ● Separate from INDEX – Sample dependent
- 43. 43 NGS: DGE Analysis & UMIs Adapted from BIOO Scientific NEXTflex® qRNA-Seq™ Description
- 44. 44 NGS: DGE Analysis & UMIs Screenshot of Molecular Index Plug-In (MIPI) Output Report
- 45. 45 NGS: DGE Analysis & UMIs Screenshot of Molecular Index Plug-In (MIPI) Output Files
- 46. 46 Robotics: Bead Agitator vs. Liquid Handling ● Maxwell 16 – Promega – Closed Source / Kits ● BiomekFX – Beckman Coulter – Open Platform or Kits – 96 / 384 wells
- 47. 47 Robotics: Bead Agitator vs. Liquid Handling ● Maxwell 16 – Promega – Closed Source / Kits – simplyRNA LEV tissue kit ● $264.60 per kit ● $5.52 per sample ● DNA from: – Plant, Animal Tissue, FFPE, Virus, Mouse Tail, Blood, Buccal Swab ● RNA from same ● HIS-tagged Protein ● BiomekFX – Beckman Coulter – Open Platform or Kits – 96 / 384 wells ● Kits for just about everything and anything – Many companies have divisions that just help you automate their reagents.
- 48. 48 Fragment Recovery – SAGE Pippin Prep ● Automated DNA fragment recovery – 50 bp ~ 1500 bp ● Can recover from 4 samples ● Sequence compatible bufer ● $50 per cassette / $12.5 per sample
- 49. 49 Controlled Fragmentation: Covaris M220 Ultrasonicator ● Temperature controlled via Peltier chiller unit – Consistent thermal control ● Significant advantages over enzymatic shearing – no sequence bias – truly random fragmentation – high recovery – highly reproducible
- 50. 50 High Performance Computing Options ● RedHawk HPC Cluster – On campus, fast, easy to use, well-supported – Limited accessibility, limited size, older components ● Ohio Supercomputer Center (OSC) – Columbus (transfer is slower) – Extremely FAST, very LARGE – Less well-supported – Need to write a proposal to justify usage ● 5 ~ 7 pages + references → outside peer review ● 10,000 ~ 30,000 RUs (up to 300,000 CPU hours (i.e. man-hours)
- 51. 51 High Performance Computing Options ● Amazon Web Services – Virtual Computers – Pay as you go (per hour) – Elastic Computing – AWS Genomics Group ● Focussed on Health Care ● Google Web Services – Virtual Computers – Pay as you go (per min) – Elastic Computing – Supported by ONIX ● Genomics Consultants
- 52. 52 Oxford Nanopore Technologies: minION
- 53. 53 ONT: minION – How does it work?
- 54. 54 ONT: DNA Sequencing Kits
- 55. 55 ONT: What to do with the data? ● Many open source bioinformatics sofware projects
- 56. 56 Long-Read Assemblers → “dirty” data ● Data quality much poorer than Illumina or Sanger ● The confidence of each base call is lower ● The Celera Assembler was well suited too this – Developed during the high-throughput days of the human genome project to build the WGS contigs – Open sourced in 2004 ● Adapted for 454 assembly ● Adapted for PacBio & Oxford Nanopore ● Now ‘forked’ and is called CANU
- 57. 57 CANU Assembler ● CANU @github ● Extremely extensive documentation ● http://canu.readthedocs.io/en/latest/ http://www.genome.org/cgi/doi/10.1101/gr.215087.116 Genome Research
- 58. 58 CANU Assembler: Three Stages; Iterative Assembly; Fully Automated; ‘Biologist’ Friendly ● Correction – Most intensive disc space ● Trim – CPU heavy; Disc space reduced ● Assembly – CPU Intensive CANU Requires a ‘CLUSTER’ or ‘Super Computer’
- 59. 59 Graduate of MiamiU MBI MSc Programme https://www.nasa.gov/mission_pages/station/research/experiments/2181.html
- 60. 60 minION: International Space Station (ISS) NASA Astronaut Kate Rubins sequenced DNA in space for the first time ever for the Biomolecule Sequencer investigation, using the minION sequencing device. Courtesy of NASA / ISS
- 61. 61 minION: How “good” is the data? 15 seconds to Identification from DATA Acquisition
- 62. 62
- 63. 63 NGS: Long-Read Technologies ● ONT minION ● $1000 instrument + Computer (~$1,500) ● Starter pack ● $200 ~ $400 per run ● 10 minute prep RAPID protocol ● 15 GB DATA output (max) ● PacBio Sequel ● $350,000 Instrument ● $850 per run ● 10 GB DATA output 3 ft 5 ft 800 lbs
- 64. 64 Acknowledgments ● Miami University ● College of Arts & Sciences ● Departments – Biochemistry & Chemistry – Biology – Microbiology ● User Community