Learning Goals

  1. Compare major sequencing platforms and approaches
  2. Examine major data formats for DNA sequencing
  3. Analyze sequence data from the command line

Introduction

  • Colautti Lab research overview

FIRST Generation Sequencing

a.k.a. Sanger Sequencing

  • Sanger overview
  • DNA Barcodes (e.g. Barcode of Life Database BoLD)
  • Quick overview of extraction & PCR protocol
  • DNA barcodes tutorial

Classic sequencing uses the Sanger method (Wikipedia).

Step 1. Extract and Purify DNA

Step 2. Select single target (e.g. PCR)

Step 3. Dye-terminator PCR

Step 4. Visualize on a gel

Key points

  • Sanger sequencing is the ‘classic’ sequencing method
  • Gel can read only one target sequence at a time
  • DNA barcodes can be used to identify species
  • R can be a powerful interface for visualizing & analyzing sequencing data

SECOND Generation

  • Overview of sequencing methods
  • Overview of de novo assembly
  • de novo assembly tutorial
  • Overview of transcriptome analysis
  • Differential expression tutorial
  • Metagenomics overview

Typical Workflow

Illumina Sequencing Overview

Key points

  • There are several flavours of ‘next generation’ sequencing (NGS)
  • NGS platforms sequence many fragments simultaneously, unlike the Sanger method
  • Sequencing technology is evolving faster than Moore’s Law of computation
  • Dealing with billions to trillions of base pairs of data is not trivial
  • Bioinformatics is usually the bottleneck and main cost of a project involving NGS

THIRD Generation

Nanopore MinION metagenomics

What’s in My Pot?

  • Inspect the data on the One Codex website
  • Can be run in ‘real time’ (i.e. BLAST results as they are sequenced)

Sequencing Comparison Table

Platform Instrument Mreads Length Gbp Type
Illumina NovaSeq 6000 S4 10,000 300 3000.00 SR or PE
Illumina NovaSeq 6000 S3 6,600 300 1980.00 SR or PE
Illumina NovaSeq 5000/6000 S2 3,300 300 990.00 SR or PE
Illumina NovaSeq 5000/6000 S1 1,600 300 480.00 SR or PE
Illumina NextSeq 500 High-Output 400 300 120.00 SR or PE
Illumina HiSeq X 375 300 113.00 PE
Illumina HiSeq 3000/4000 313 300 93.80 SR or PE
Illumina NextSeq 500 Mid-Output 130 300 39.00 PE
Illumina HiSeq High-Output v4 250 250 62.50 SR or PE
Illumina HiSeq High-Output v3 186 250 46.50 SR or PE
Illumina HiSeq Rapid run v4 150 500 75.00 SR or PE
Illumina HiSeq Rapid Run 151 300 45.20 SR or PE
Illumina HiScanSQ 93 200 18.60 SR or PE
Illumina GAIIx 42 300 12.60 SR or PE
Illumina MiSeq v3 25 600 15.00 SR or PE
Illumina MiniSeq High-Output 25 300 7.50 SR or PE
Illumina MiSeq v2 16 250 4.00 SR or PE
Illumina MiniSeq Mid-Output 8 300 2.40 SR or PE
Illumina MiSeq v2 Micro 4 300 1.20 SR or PE
Illumina MiSeq v2 Nano 1 500 0.50 SR or PE
Ion Proton I 60 200 12.00 SR
Ion PGM 318 4 400 1.60 SR
Ion PGM 316 2 400 0.80 SR
Ion PGM 314 0.4 400 0.16 SR
Roche 454 GS FLX+ / FLX 1 700 0.49 SR
Roche 454 GS FLX+ / FLX 0.35 700 0.24 SR
Roche 454 GS FLX+ / FLX 0.13 700 0.09 SR
Roche 454 GS FLX+ / FLX 0.05 700 0.04 SR
Roche 454 GS FLX+ / FLX 0.02 700 0.01 SR
Roche 454 GS FLX+ / FLX 0.07 400 0.03 SR
SOLiD 5500xl W 267 100 26.70 SR or PE
SOLiD 5500 W 267 100 26.70 SR or PE
SOLiD 5500 82 100 8.15 SR or PE
SOLiD 5500xl 82 100 8.15 SR or PE
PacBio PacBio Sequel 0.37 20,000 7.40 SR
PacBio PacBio RS II (P6) 0.06 15,000 0.80 SR
Oxford Nanopore MinION ?? 2,000,000 20.00 SR
Oxford Nanopore PromethION ?? 2,000,000 1000.00 SR