Presented By: Emily Lamoureux Microbial Sequencing Presented By: Emily Lamoureux

16 rRNA sequencing Taxonomic analysis of bacteria in a community

4. Bioinformatic Pipeline 1. DNA extraction PowerFecal MO BIO 3. Illumina MiSeq 2. Barcoded PCR V6 – V8 16S DNA 4. Bioinformatic Pipeline https://github.com/mlangill/microbiome_helper/wiki Extract DNA, amplify part of 16S gene via PCR, also add sample specific barcodes and primers for sequencing platform.

16S rRNA Sequencing C8 C9 V6 V8 V7 Bacterial Taxa PCR Bacteroidales Clostridiales PCR barcode adapters C8 C9 300 bp 16S rRNA sequencing leverages the variable regions in the 16S rRNA gene, which is present bacteria. These variable regions are unique to each bacterial species, providing a signature that we can use to identify the bacterial composition of a sample. Primers are designed for the more conserved regions that interspace the variable regions, and then each sample is amplified with specific barcodes, so they can later be identified from the pooled sequenced samples. Once you have your sequencing results, you can identify a bacterial profile for each sample – two common orders of bacteria found in the gut - have a break down of genus and hopefully species after that. Before going into microbiome data ~ 438 bp Stich paired reads

5’ 3’ Nextera P5 adapter Nextera P7 adapter Nextera XT v2 i5 indices S5xx “insert” sequence i7 N7xx 5’ 3’ left arm right arm F R right arm left arm 29 nt 8 nt 33 nt 17 nt 17 nt 34 nt 8 nt 24 nt ideally ≈ 450 nt Nextera P5 adapter Nextera P7 adapter Nextera XT v2 i5 indices Nextera XT v2 i7 indices Target-specific forward primer Target-specific reverse primer Metagenomics – NexteraXT Transposase enzyme carries the right arm of the illumina adapter – cuts and ligates Then PCR to put on left arm and barcode

Beta-diversity plot Skunk Marten Mink Bear Otter Fisher Snowshoe hare Groundhog Domestic rabbit Raccoon Porcupine Beaver Moose Elk Red Deer Swift Fox Red Fox Arctic Fox Coyote Arctic Wolf Timber Wolf Labels: Starting top left Caramel – 6 (Skunk) Bright yellow – 14 (Marten) Pale yellow – 5 (Mink) Light mint – 3 (Otter) Bright pink – 8 (Bear) Orange – 11(Fisher) Purple – 13 (Snowshoe Hare) Tan – 22 (Ground hog) Dark grey – 2 (Domestic Rabbit) Light grey – 7 (Racoon) Sky blue – 9 (Porcupine) Pale blue – 16 (Beaver) Peach – 20 (Moose) Dark blue – 10 (Elk) Brown – 19 (Red deer) Light pink – 17 (Swift Fox) Red – 1 (Red Fox) Green – 12 (Arctic Fox) Sea green – 18 (arctic wolf) Pale purple – 4 (coyote) Pastel blue – 21(Timber wolf)

Shotgun metagenomic sequencing Taxonomic analysis of bacteria in a community

2. Genome fragmentation & PCR (transposase) 4. Bioinformatic Pipeline 1. DNA extraction PowerFecal MO BIO 3. Illumina NextSeq 2. Genome fragmentation & PCR (transposase) 4. Bioinformatic Pipeline https://github.com/mlangill/microbiome_helper/wiki Extract DNA, amplify part of 16S gene via PCR, also add sample specific barcodes and primers for sequencing platform.

http://teachthemicrobiome.weebly.com/sequencing-the-microbiome.html

Supplementary

Illumina Miseq Technology https://www.youtube.com/watch?v=womKfikWlxM http://cgeb-imr.ca/index.html Integrated Microbiome Resource (IMR)

Command line tutorial Unix and Perl for Biologists: http://korflab.ucdavis.edu/Unix_and_Perl/index.html

NOTE Keep track of everything: data you input commands you run analyses you do (including relevant parameters if someone were to repeat it) Just like you would write down protocols in your lab book (though its easier to just put a README.txt in your working folder