1. Introduction to RAD
Acropora millepora

2. First thing start downloading course achieves
See downloading course archives

3. Plan Start downloading and unpacking data Set up our reference genome
Go through some introduction slides
Go through GATK demonstration files
User choice, read preparation, de novo, or GATK for real

4. Genetic differentiation vs. distance
Population Genetics N S O M K W A
R2 = 0.76
Genetic differentiation vs. distance

5. Linkage Mapping
Final map consists of 3816 markers and covers 99.4% (1539cM) of scallop genome with a resolution of 0.41cM.

6. 2bRAD-based linkage mapping in scallop
sex-related chromosomal region
Based on the high-density map, we were able to identify a 2-cM chromosomal region that contained ~60 sex-related loci.

7. Which loci are under selection across populations?
(Restriction-Associated DNA, RAD)
Generate and sequence short tags randomly distributed across genome
Fst along linkage group III: between freshwater populations (orange) and between freshwater and marine populations (black). Bars: significant (bootstrap), dots: SNPs

8. Population genomics: “genome scanning” for signatures of selection
Nucleotide diversity (recent selection)
Tajima’s D
(not so recent selection)
coalescent
hard sw. LD2hs neutr balancing neutr soft sw.
(recent or continuous selection)
Excess LD
(very recent selection)
position along genome
Hohenlohe et al 2010 Int. J. Plant Sci. 17:

9. Amplification and barcoding
Adaptor ligation
and indexing
III
Index (1 of 12)
(mix indices by 12)
Amplification and barcoding
Barcode2
III
Gel cleanup
Gel cleanup
III

10. Restriction Digest Type IIb restriction enzyme is used to cut out fragments of the genome
genomic DNA:
BcgI cut site
5’- NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN-3’
digested fragments represent a random subset of the genome
3’-NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN -5’

11. Ligation Step Adapters are ligated to the restriction fragments produced by the digest
Adapter 1: contains degenerate bases to ID PCR duplicates
5Ill-NNRW + anti 5Ill-NNRW
5’-CTACACGACGCTCTTCCGATCTNNRWCCNN-3’
5’-GGWYNNAGATCGGAAGAGC/3InvdT1/-3’
Adapter 2: Contains a 3’ barcode (in red below)
3Ill-BC[1-12] + anti 3Ill-BC[1-12]
5’-CAGACGTGTGCTCTTCCGATCTACCANN-3’
5’-TGGTAGATCGGA/3InvdT/
5’- NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN-3’
3’-NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN -5’
5’CTACACGACGCTCTTCCGATCTNNRWCCNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
TGGTAGATCGGA/3InvdT/
/3InvdT1/CGAGAAGGCTAGANNYWGG
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’

12. Using degenerate bases to identify PCR duplicates
Adapter 1:
5Ill-NNRW + anti 5Ill-NNRW
5’-CTACACGACGCTCTTCCGATCTNNRWCCNN-3’
5’-GGWYNNAGATCGGAAGAGC/3InvdT1/-3’
N = A, T, G, or C
R = A or G
W = A or T
Y = C or T
5’-CTACACGACGCTCTTCCGATCTNNRWCCNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
TGGTAGATCGGA/3InvdT/
/3InvdT1/CGAGAAGGCTAGANNYWGG
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’
Degenerate bases included in the 5Ill-NNRW adapter

13. Amplification (perform on pooled samples)
Ill-[1-12]-bc
CAAGCAGAAGACGGCATACGAGAT[barcode]GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
Mpx2N
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT
IC1-P5
AATGATACGGCGACCACCGA
IC2-P7
CAAGCAGAAGACGGCATACGA
Mpx2N
Ill-bc
AATGATACGGCGACCACCGA
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT
5’-CTACACGACGCTCTTCCGATCTNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
TGGTAGATCGGA/3InvdT/
/3InvdT1/CGAGAAGGCTAGA
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’
TAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG[barcode]TAGAGCATACGGCAGAAGACGAAC
AGCATACGGCAGAAGACGAAC

14. Amplification Ill-[1-12]-bc
CAAGCAGAAGACGGCATACGAGAT[2ndbarcode]GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
Mpx2N
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT
IC1-P5
AATGATACGGCGACCACCGA
IC2-P7
CAAGCAGAAGACGGCATACGA P5 P7
AATGATACGGCGACCACCGA
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT
5’-CTACACGACGCTCTTCCGATCTNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
TGGTAGATCGGA/3InvdT/
/3InvdT1/CGAGAAGGCTAGA
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’
TAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG[barcode]TAGAGCATACGGCAGAAGACGAAC
AGCATACGGCAGAAGACGAAC

15. 2nd barcode was added during PCR
Final Product p5 p7
Read primer
sampled genomic DNA
1st barcode
ligated onto fragment
2nd barcode was added during PCR
5’-CTACACGACGCTCTTCCGATCTNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
/3InvdT1/CGAGAAGGCTAGA
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’
TGGTAGATCGGA/3InvdT/
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT
AATGATACGGCGACCACCGA
TAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG[barcode]TAGAGCATACGGCAGAAGACGAAC
AGCATACGGCAGAAGACGAAC

16. Double Barcoding p5 p7 sampled genomic DNA Ligation indexes
Read primer p5 p7
random bases for duplicate identification
sampled genomic DNA
1st barcode
ligated onto fragment
2nd barcode was added during PCR
Ligation indexes
PCR barcodes
Setting unique ligation barcodes to columns and PCR barcodes to rows gives a unique combination for each sample A1 B1
If groups using strip tubes, each strips will be layed down as rows in this picture. Then each tube in the strip gets a different adapter barcode and the whole strip can be pooled downstream.

17. Pooling Samples
Pooling allows for 12 uniquely barcoded samples to be prepared in a single tube.
Saves work and pipet tips
Use different Adapter 2 for each column (1-12)
Pool samples so that each sample in the pool has a different ligation adapter
5’-CTACACGACGCTCTTCCGATCTNN
NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
TGGTAGATCGGA/3InvdT/
/3InvdT1/CGAGAAGGCTAGA
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN
NNACCATCTAGCCTTCTCGTGTGCAGAC-5’
Pooled sample 1
If groups using strip tubes, each strips will be layed down as rows in this picture. Then each tube in the strip gets a different adapter barcode and the whole strip can be pooled downstream.

18. GATK

19. Genome reference pipeline (GATK) (http://www. broadinstitute
Trimming/quality filtering
Mapping to genome
Realign around indels
Primary variant calling
Base quality recalibration
Secondary variant calling
Variant quality recalibration
based on genotyping replicates
Final filtering
Assess quality (heterozygote discovery rate)

20. Genetic differentiation vs. distance
Acropora millepora connectivity along the Great Barrier Reef
R2 = 0.76
Genetic differentiation vs. distance N S O M K

21. SAM File Format Header Lines @HD:
Header line (first line of file if present)
gives the version number and sorting information, here version 1, unsorted alignments.
@SQ:
These lines give the reference sequence information. There will be as many of these as ‘chromosomes’ in your reference. In our case this is 10. Gives the name of the sequence and its length.
@PG:
Program line. Gives information about the program used to perform the alignment. In our case bowtie2 version

22. SAM File Format Alignment Lines example from our files
form ‘Sequence Alginment/Map Format Specifications’—The SAM/BAM Format Specification Working Group 2015 (see under additional resources)

23. VCF file Structure VCF = variant call format
from ‘The Variant Call Format Specifications’ 2015

24. VCF file Structure VCF = variant call format
1 row for each variant position
columns give individual sample data
Fields that describe the variant
Sample Data
This individual is a heterozygote G/T
With a GQ score of 21
Read depth of 6
Haplotype quality of 23, 27
Descriptions of what these these mean are included in the header (see previous slide)
from ‘The Variant Call Format Specifications’ 2015

25. Recalibration Plots

26. Recalibration Plots

27. Recalibration Plots

29. Writing your own pipeline