Teagasc/APC Sequencing Facility Fiona Crispie and Laura Finnegan Position Fiona.crispie@teagasc.ie

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

Sequencing Facilities at Moorepark

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 v2 15 M 50, 300, 500 7.5 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)

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

Illumina Sequencing

Illumina Sequencing

Illumina Sequencing

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

Ion PGM and Proton

Oxford Nanopore MinIon

Nanopore sequencing

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

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

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

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

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!

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.

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”)

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

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

Questions/Quotations/ Grant costings Contact: Dr. Paul Cotter (paul.cotter@teagasc.ie) Or Dr. Fiona Crispie (fiona.crispie@teagasc.ie)

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