1. Sequencing technology and assembly1

2. Sanger sequencing Sanger sequencing with radioactivity
High throughput Sanger sequencing with fluorescence 2

3. Roche/454 sequencing Yield: 500,000,000 bp Cost: $5,000
Time: ~1 min per bp
Read length: 450 bp - > 1kb

4. Pyrosequencing

5. Illumina sequencing Yield: 8,000,000,000 – 80,000,000,000 bp
Time: ~1 hour per bp
Read length: ~150 bp
Cost:
Sample Extraction, $14.00/sample
Automated Sample Library, $90.00/sample
MiSeq (2x250), 1 lane 8-10Gb/lane, $1,700.00/sample
MiSeq (2x300), 1 lane, 10-12Gb/lane, $2,100.00/sample
HiSeq2500 (2x150), 1 lane, ~40Gb/lane, $2,500.00/lane
HiSeq2500 (2x250), 1 lane, ~65Gb/lane, $3,500.00/lane 5

6. Illumina sequencing

7. Ion Torrent Yield: 50,000,000 bp Time: 2 hours Read length: 500bp
<1 min per bp
Cost: $500 7

8. Ion Torrent

9. PacBio Long reads (5-10kb) High error, but read 150x coverage
Library prep: $600
Sequencing: $300

10. PacBio

11. Minion
Quick sample prep
Long reads (~50kb)
High error
$150 per run

12. Minion

13. Errors
Different technologies have different error rates:

14. Base calling Need to be sure which base you have identified
Depends on the technology
Each machine includes software
Phred is an historical package developed by at U. Washington
Phred scores are probability that the base is correct 14

15. Quality values Phred 10: 1 x 101 chance that the base is wrong
Phred 99: the base is correct!
Fastq scores are the score + 33 then converted to ascii text 15

16. Homopolymeric errors Homopolymeric runs: Signal is not linear
Not clear if 5 or 6 bases

17. Errors Different technologies have different error rates:
Pyrosequencing/Ion Torrent – homopolymeric tracts
Illumina – substitution errors
PacBio – Machines can not keep up with biology
Minion – noise coming through the membrane