1. Presented By: Emily Lamoureux
Microbial Sequencing
Presented By: Emily Lamoureux

2. 16 rRNA sequencing
Taxonomic analysis of bacteria in a community

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

4. 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. 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

6. 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)

7. Shotgun metagenomic sequencing
Taxonomic analysis of bacteria in a community

8. 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
Extract DNA, amplify part of 16S gene via PCR, also add sample specific barcodes and primers for sequencing platform.

10. Supplementary

11. Illumina Miseq Technology
Integrated Microbiome Resource (IMR)

12. Command line tutorial
Unix and Perl for Biologists:

13. 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