1. Today HK WHY sequence? Minion library prep Minion sequencing run *.ppt
Bioinformatics MITO, primer walking
Fungal samples

3. SNAIL AND PARASITES BIOLOGY
DNA
“identity, possibilities”
phylogenetics
CTAB/DNAzol
CTAB/DNAzol
gel electrophoresis
nanodrop spec
Illumina (full)
genome sequencing
PCR rDNA/mito
Qubit Fluorometry
Covaris fragmentation
Ampure (fragment collection)
Kapa DNA library preparation kit
Pippin size selection
QC Bioanalyzer, Qubit, qPCR
Illumina run
TA cloning, B/W screening
electrophoresis
Qiagen plasmid extraction Restriction digests
direct sequencing
M13 sequencing
Sequence ID (BLAST)
editing
Primer design, walking
Galaxy
QC Data file
(MT) genome assembly
Mitos, manual annotation
Gene annotation
Phylogenetics
GenBank submission

4. FREPS + SNAIL PLASMA LECTINS THAT ARE PARASITE ANTIGEN- REACTIVE
GENOMIC DNA C SP
IgSF1
SCR
IgSF2
ICR
FBG N
PROTEIN
IgSF
FIBRINOGEN-RELATED PROTEINS (FREPs) +
parasite
Biomphalaria glabrata
FREPS are snail plasma lectins that are parasite-antigen reactive. Once the snail Biomphalaria glabrata is infected with a parasite like the trematode Echinostoma paraensei, a precipitate occurs in the blood of the snail. The precipitate consists of parasite antigens and snail plasma lectins. These lectins include fibrinogen-related proteins or FREPS. There are two types of FREP genes. Shown are the structure for the proteins and the genes. Freps have a namesake downstream fibrinogen-like domain and upstream immunoglobulin superfamily domains, either one or two.
IMMUNOGLOBULIN SUPERFAMILY
(IgSF)-LIKE DOMAIN
FIBRINOGEN b/g
(FBG)-LIKE DOMAIN
UNIQUE COMBINATION
precipitate

5. FREPS + SNAIL PLASMA LECTINS THAT ARE PARASITE ANTIGEN- REACTIVE
THAT ARE DIVERSIFIED SOMATIC MUTATIONS A B +
parasite
Biomphalaria glabrata
GENOMIC DNA C SP
IgSF1
SCR
IgSF2
ICR
FBG N
PROTEIN
IgSF
FIBRINOGEN-RELATED PROTEINS (FREPs)
GENOMIC PCR AMPLICONS CLONED AND SEQUENCED
NT LEVEL CLUSTERS
AA LEVEL CLUSTERS
FREPS are snail plasma lectins that are parasite-antigen reactive. Once the snail Biomphalaria glabrata is infected with a parasite like the trematode Echinostoma paraensei, a precipitate occurs in the blood of the snail. The precipitate consists of parasite antigens and snail plasma lectins. These lectins include fibrinogen-related proteins or FREPS. There are two types of FREP genes. Shown are the structure for the proteins and the genes. Freps have a namesake downstream fibrinogen-like domain and upstream immunoglobulin superfamily domains, either one or two.
Zhang, Adema, Kepler, Loker 2004 5

6. SOMATIC DIVERSIFICATION POINT MUTATION AND GENE CONVERSION
FREP3 DIVERSITY
Zhang et al 2004
FREP3
GENOMIC DNA
(375 BP) C SP
IgSF1
SCR
IgSF2
ICR
FBG N
PROTEIN
nucleotide position
330 50
100
150
200
250
300
103 90
124 92
122 94 98 12
117
120
112
118
FREP3 variant
140
141
147
148
155
158
165
175 13
177
164 A B
Analysis of FREP3, specifically the exon that encodes an IgSF domain, yielded many highly diverse PCR-amplified sequences from whole body DNA of individual snails like A and B shown here. These blocks represent the mosaic sequence variants of FREP3 for each snail. Colors indicate sections of sequence that derive from separate loci, white dots indicate unique SNPs. This sequence diversity indicates somatic diversification, the patterns observed are best explained by point mutations and gene conversion. Gene conversion is a nonsynonymous reciprocal recombination process that results in one region of DNA becoming identical with another, .
SOMATIC DIVERSIFICATION POINT MUTATION AND GENE CONVERSION

7. FREP gene structure and estimated number
Clustering of FREP genes in genome, Assembly?

8. Biomphalaria glabrata “FINAL” ASSEMBLY STATISTICS
ASSEMBLY: ~ 916Mb (10% gaps)
Coverage ~78.5x
GENOME SIZE ~930Mb
(estimate Gregory, 2002)
N50 scaffolds: 3,051
L50 scaffolds: 36,793 nt
Predicted (Aug 2017) protein-coding genes : 25,550
CEGMA (Core Eukaryote Gene Mapping Approach)
Sequence alignments to 458 highly conserved proteins from Clustered Orthologous Groups, or KOGS, from several species to (..) assess the completeness of an assembly. Parra G et al. 2007
442 (out of 458) CEGs
96.5%
(IMPROVEMENTS, UPDATES ARE ONGOING, WELCOME)

10. GENOME ORGANIZATION FREPs
67 clones from a B. glabrata BAC library strongly hybridized FREP gene-specific probes, frequently binding multiple probes.
FREP 2
FREP 7
FREP 3
FREP 12
One IgSF domain
Two IgSF domains 1 2 3 4 5 6 7 8 9 10 11 12 A B F E D C
FREP 3
FREP 12
FREP 7
FREP 2
FREPs cluster in B. glabrata genome

11. 120,000 bp
Repetitive content
GAPS after combined effort
UNM plus
Genomics Center WashU

12. TANDEM ARRANGEMENT OF FREP GENES, FRAGMENTS
BIOMPHALARIA GENOME
TANDEM ARRANGEMENT OF FREP GENES, FRAGMENTS
GenBank JN (also see AC235916), multiple scaffolds in current genome assembly
ASSEMBLY FROM SHORT READS CONTINUES TO BE CHALLENGING

13. In Genbank: still a question regarding genomic organization and clustering

14. Linearizing and fragmenting BACs, what enzyme to use?
“4 FREPs 2 gaps”
058F09
”1FREP, fragments and 1 gap”

15. POOL to ~1mg Autorads from 204
0021H07
0056M13
0058F09
0075L08
0090M06
0091G14
0096N07
0125N01
Autorads from 204
Identify the following BAC clones as FREP probe reactive:
# F2 F3 F4 F7 F12 F13
0003D X
0009K X
0011G01 X X
0012C17 X X X
0020E24 X X
0020M13 X X X
0021E X
0021H07 X
0030E X
0031E X
0033H X
0056M13 X X X
0058F09 X
0067K X
0075L08 X
0070P X
0071J X
0071P X
0090M06 X
0091G14 X X
0096N07 X
0105H12 X
0121H09 X X X
0125N01 X X
0141F X
0142D X
0150H04 X X
0153J15 X X
0153N X
0155N05 X
0157F X
POOL to ~1mg

20. *.ppt

21. Sequences available from primer walking:
Group reaction 1 reaction 2
plasmid primer plasmid primer

22.
Genetic code 5 vs 9