1. Teagasc/APC Sequencing Facility
Fiona Crispie and Laura Finnegan
Position

2. What we have
Illumina NextSeq
Illumina MiSeq
MinIon
Ion PGM
Ion Proton

3. Sequencing Facilities at Moorepark

4. Illumina MiSeq Specifications: MiSeq Reagent Kit No. of Reads
Kit Size (cycles)
Output (max.)
MiSeq Reagent Kit v3
25 M
150, 600
15 Gb
MiSeq Reagent Kit v M , 300, Gb
Examples of Projects
16S Compositional Sequencing
Shotgun Metagenomic
Genome (bacterial and phage de novo and resequencing)
Virome
Fungal populations
Evolution of fungal pathogen genes (SNP variant detection)

5. Illumina NextSeq Specifications: Examples of Projects
NextSeq 550 System High-Output Kit*
NextSeq 550 System Mid-Output Kit*
Read Length
Output
2 × 150 bp
100–120 Gb
32.5–39 Gb
2 × 75 bp
50–60 Gb
16.25–19.5 Gb
1 × 75 bp
25–30 Gb
Single Reads
Up to 400 Million
Up to 130 Million
Paired-End Reads
Up to 800 Million
Up to 260 Million
Examples of Projects
Shotgun Metagenomic
ATAC libraries
RNAseq

6. Illumina Sequencing

7. Illumina Sequencing

8. Illumina Sequencing

9. Ion PGM and Proton Ion PGM™ Chip Output 200 bp read 400 bp read
Ion 314™ Chip v2
30–50 Mb
60–100 Mb
Ion 316™ Chip v2
300–500 Mb
600 Mb–1 Gb
Ion 318™ Chip v2
1.2–2 Gb
Ion Proton™ Chip
Ion PI™ Chip
Up to 10 Gb

10. Ion PGM and Proton

11. Oxford Nanopore MinIon

12. Nanopore sequencing

13. Sequencing! What do we do? DNA/RNA extraction: Library Preparation:
QC:
Agilent and qPCR

14. DNA extraction
Faeces
Organs such caecum, small intestine, lung, bladder…
Rumen contents

15. DNA extraction Hindgut of ticks! Food (cheese, milk, yogurt, kefir)
Soil

16. If aliquoting and freezing, ideally aliquot into tubes with beads (if using)
Omnigene tubes good for fresh samples
Do a negative extraction control- especially for samples with expected low diversity/low bacterial load…or when trying out new kits….
RNAlater for samples for RNA extraction

17. Library Preparation DNA requirements:
Resuspend in Tris-Cl pH7.5-8 or water (No TE)
Amplicon (Illumina): 5ng/ul DNA
Shotgun (Illumina): 1ng DNA – 5 ul of 0.2ng/ul
Amplicon (Ion): 100pM for enrichment
Shotgun (Ion): Minimum 100ng prior to fragmentation, 100pM post fragmentation
16S (MinIon): 10ng for amplification
Shotgun (MinIon Rapid prep): 400 ng!

18. Library Preparation RNA requirements:
The RNA sample should be free of salts (e.g., Mg2+ or guanidinium salts) or organics (e.g., phenol and ethanol) and have RIN values of 8-10.
Eukaryotic: Preferably at least 500ng in 5ul
Prokaryotic: 1ug total RNA for rRNA depletion, then >/= 50ng rRNA depleted, DNA – free RNA.

19. As well as negative extraction controls, include negative PCR controls (set up negative control last)
Keep pre and post-PCR areas separate, use UV hoods where possible
Observe GLP
Index negative controls with completely unique indices (eliminates risk of “index swopping”)

20. Types of libraries sequenced
16S metagenomic: (MiSEq, PGM, Proton, MinIon (full length))
Shotgun metagenomic and genomic sequencing: (MiSeq, PGM, Proton, NextSEq, MinIon)
SNP: (PGM)
TRADIS: MiSeq
ATAC–libraries
RNAseq

21. Method development: (Also known as “in our spare time”!)
Shotgun kit comparison study: Comparing outputs from Nextera XT, Nextera Flex, kits from Kapa and NEB
Comparison of RNA extraction methods from foods
RNAseq on microbial transcriptome- comparison of Illumina Script Sense kit with methods involving adding poly A tails to transcripts

22. Questions/Quotations/ Grant costings
Contact:
Dr. Paul Cotter Or
Dr. Fiona Crispie

23. Dr. Paul Cotter Laura Finnegan Vision 1 Team
Acknowledgements
Dr. Paul Cotter
Laura Finnegan
Vision 1 Team