Figures and data in Copy number variation and population-specific immune genes in the model vertebrate zebrafish

Figures
Tables
Additional files

4 figures, 6 tables and 1 additional file

Figures

Figure 1

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Structure of zebrafish NLRs and a map showing the origin of wild zebrafish samples.

(A) Generalized, schematic representation of the domain architecture of an NLR-C protein. Each box represents a translated exon. The N-terminal repeats, the death-fold domain, as well as the B30.2 domain only occur in subsets of NLR-C genes. The number of N-terminal repeats and leucine-rich repeats can vary. Domains that can be either present or absent in different NLRs are surrounded by square brackets. (B) Sampling sites for wild zebrafish. All sites are located near the Bay of Bengal. Final sequenced sample sizes are indicated in parentheses. The map is based on geographic data collected and published by AQUASTAT from the Food and Agriculture Organization of the United Nations (FAO, 2021). The population DP is marked with an asterisk because its analysis and results are presented only in figure supplements.

Figure 2 with 3 supplements

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Total counts of NLRs found per individual, shown for each population.

Black diamonds on the box plots denote means, horizontal lines denote medians. Left side: two laboratory strains; right side: three wild populations.

Figure 2—source data 1 Source tables for Figure 2 and its supplements.: https://cdn.elifesciences.org/articles/98058/elife-98058-fig2-data1-v2.xlsx
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Figure 2—source data 2 Sequences and target locations of RNA baits.: https://cdn.elifesciences.org/articles/98058/elife-98058-fig2-data2-v2.xlsx
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Figure 2—figure supplement 1

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Sequencing and assembly statistics of circular consensus sequence (CCS) reads from NLR exons.

(A1) Absolute numbers of CCS reads from NLR exons per sequenced individual. (A2) Lengths of the CCS reads that map to NLR genes. (B1) Absolute numbers of assembled contigs containing an NLR exon per sequenced individual. Triangles above TU mark the numbers of NLR exons found in the reference genome. (B2) Lengths of the individual assembled contigs that contain an NLR exon. Outliers not shown in the boxplots. The black diamonds on boxplots denote means, horizontal black lines denote medians.

Figure 2—figure supplement 2

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Assembled NLRs in the reference genome GRCz11.

(A) Proportions of unique FISNA-NACHT and NLR-B30.2 sequences that were successfully mapped to the reference genome GRCz11 with a mapping quality of 60, by population. (B) Distribution of mapping qualities for all unique NLR sequences that aligned to GRCz11, showing that most map either with very high (60) or very low quality.

Figure 2—figure supplement 3

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Identification of B30.2 domains associated with zebrafish NLRs.

(A) Nucleotide sequence logo (on top, continued on bottom left) and amino acid logo (on bottom right) for a small, highly conserved 47 bp exon that precedes B30.2 in zebrafish NLRs and not in other genes. The first nucleotide of the exon was removed to generate the correct amino acid translation. Logos were created with Weblogo, the height of each base represents its information content in bits (Crooks et al., 2004). (B) Absolute numbers of contigs containing a B30.2 exon per sequenced individual, split by presence/absence of the NLR-specific exon. The black diamonds on boxplots mark the means. (C) Genomic distribution of FISNA-NACHT domains in the GRCz11 reference genome. (D) Genomic distribution of B30.2 domains in the GRCz11 reference genome.

Figure 3 with 2 supplements

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Copy number variation of NLR genes.

(A) Sequence data from each individual zebrafish (vertical axis) was aligned to FISNA-NACHT exon sequences of the pan-NLRome (horizontal axis). Grayscale intensity shows, for each NLR, the proportion of NLR-aligning data in each given fish that matches this specific gene. Darker gray indicates a higher likelihood of this NLR being represented in multiple copies in the particular individual. Light gray indicates a single copy, white indicates absence. For clarity, only the 1235 FISNA-NACHT exons for which at least one fish had a minimum of 10 reads mapped to it are shown. (B) Numbers of pan-NLRome sequences (based on FISNACHT diagnosis) found in all three, two, or only one wild population. (C) Relative numbers of fish in which pan-NLRome sequences were found in wild populations. ’Core’ pan-NLRome: genes which are found in at least 80% of the sample (from a total of 57 wild fish); ’shell’: genes in at least 20%; ’cloud’: rare genes found in less than 20% of the sample. (D) Observed and estimated sizes of population-specific pan-NLRomes. Data points (filled circles and squares) show the average number of totally discovered NLR genes (as identified via their FISNA-NACHT domain) when investigating $x$ fish. The dashed line is obtained by non-linear fit of the data to the function given in Equation 2. For all populations, the hypothetical pan-NLRome size – when extrapolating $x \to \infty$ – is finite (see Table 1).

Figure 3—source data 1 Source tables for Figure 3 and its supplements.: https://cdn.elifesciences.org/articles/98058/elife-98058-fig3-data1-v2.xlsx
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Figure 3—figure supplement 1

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Comparison of copy number variation in FISNA-NACHT and NLR-B30.2 exons.

(**A1, A2**) Numbers of private and shared NLR sequences in wild populations. (**B1, B2**) Numbers of unique NLR sequences (each with one or more copies per individual) found in fish of the sequenced strains. Black diamonds on the box plots denote means, horizontal lines denote medians. (**C1, C2**) Population-specific pan-NLRomes and sets of NLR-B30.2 domains. Data points (filled circles and squares) show the average number of totally discovered NLR genes (as identified via their FISNA-NACHT domain) in $x$ individuals. The dotted lines represent the result of non-linear curve fitting (detailed in ‘Materials and methods’).

Figure 3—figure supplement 2

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Copy number variation of NLR genes, including the DP population.

(A) Sequence data from each individual zebrafish (x-axis) was aligned to FISNA-NACHT exon sequences of the pan-NLRome (y-axis). Grayscale intensity shows, for each NLR, the proportion of NLR-aligning data in each given fish that matches this specific gene. Darker colors can be interpreted as potentially having multiple copies. Lighter colors indicate a single copy, white color means that the sequence was not present. For clarity, only the 1235 FISNA-NACHT for which at least one fish had 10 reads mapped to it are shown. (B) Relative numbers of fish in which the pan-NLRome sequences were found in wild populations. Some belong to the core pan-NLRome (in at least 80% of fish), while others are classified as shell (in at least 20% of fish) or cloud (less than 20%). (C) Numbers of unique NLR sequences (each with one or more copies per individual) found in fish of the sequenced strains. Black diamonds on the box plot denote means, horizontal lines denote medians. (**D, E**) Principal component analysis of scaled-per-individual NLR (FISNA-NACHT) copy numbers. The first two components appear to separate data based on differences between wild and laboratory zebrafish (PC1), and based on geographic distance (PC2).

Figure 4 with 1 supplement

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Single-nucleotide variation in NLR exons.

Pairwise nucleotide diversity ( $θ_{π}$ ) and Watterson’s estimator of the scaled mutation rate ( $θ_{w}$ ) for FISNA-NACHT (A) and NLR-associated B30.2 (B) exons. (C) Proportion of exons without any single nucleotide polymorphisms. (D) Ratio of $θ_{π} / θ_{w}$ . Only exons with at least one single-nucleotide polymorphism are shown. The dotted, horizontal line marks a ratio of 1, the expected value under neutrality and constant population size. The black diamonds on box plots denote means, horizontal lines denote medians.

Figure 4—source data 1 Source tables for Figure 4 and its supplement.: https://cdn.elifesciences.org/articles/98058/elife-98058-fig4-data1-v2.xlsx
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Figure 4—figure supplement 1

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Single-nucleotide polymorphisms of different NLR exons shown by population, including DP.

(A) Nucleotide diversity ( $θ_{π}$ ) and Watterson estimator ( $θ_{w}$ ) for FISNA-NACHT exons. (B) Nucleotide diversity ( $θ_{π}$ ) and Watterson estimator ( $θ_{w}$ ) for NLR-associated B30.2 exons. (C) Proportion of exons which are completely monomorphic. (D) Ratio of $θ_{π} / θ_{w}$ . Only exons with at least one variant are shown. The black, dotted line marks a ratio of 1. The black diamonds on box plots denote means, horizontal lines denote medians.

Tables

Table 1

Values of fitted parameters and saturation limits for FISNA-NACHT and NLR-B30.2 exons, by population.

Population	FISNA-NACHT				NLR-B30.2
-	α	β	Limit	Quantile*	α	β	Limit	Quantile*
TU	178.274	1.43356	519.548	118	53.8579	1.40774	164.73	164
CGN	257.207	1.62786	569.367	23	78.7156	1.61283	177.246	25
DP	309.14	1.01231	25284	2930^†	69.3609	0.87454	∞	na
KG	436.761	1.2152	2288.41	2060	145.715	1.1418	1113.23	6.41e6
SN	479.892	1.26093	2152.12	3907	145.548	1.10183	1514.35	3.75e9
CHT	416.712	1.18893	2451.81	1.12e5	135.677	1.11911	1218.54	1.41e8

*

Sample size required to capture 90% of the population’s pan-NLRome.
†

DP required sample size refers to only 10% (instead of 90%) of its hypothetical pan-NLRome size.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain (Danio rerio)	Cologne zebrafish; CGN; KOLN	Other		8 Cologne fish, AG Hammerschmidt, University of Cologne
Strain (D. rerio)	Tübingen zebrafish; TU	Other		8 Tübingen fish, AG Hammerschmidt, University of Cologne
Biological sample (D. rerio)	DP	Other		20 wild fish, Dandiapalli, India (22.22155, 84.79430)
Biological sample (D. rerio)	CHT	Other		20 wild fish, Chittagong, Bangladesh (22.47400, 91.78300)
Biological sample (D. rerio)	KG	Other		20 wild fish, Leturakhal, India (22.26189 87.27881)
Biological sample (D. rerio)	SN	Other		20 wild fish, Santoshpur, India (22.93765 88.55311)
Sequence-based reagent	Baits; RNA baits; hybridization baits	Daicel Arbor Biosciences	Cat# Mybaits-1-24	Sequences available in Figure 2—source data 2
Commercial assay or kit	MagAttract HMW DNA Kit	QIAGEN	Cat# 67563
Commercial assay or kit	NucleoSpin Tissue Kit	MACHEREY-NAGEL	Cat# 740952.50
Commercial assay or kit	NEBNext Ultra II DNA Library Prep Kit	New England Biolabs	Cat# E7645L
Sequence-based reagent	NEBNext Multiplex Oligos for Illumina	New England Biolabs	Cat# E7335L	Index Primers Set 1
Commercial assay or kit	Kapa HiFi Hotstart Readymix	Kapa Biosystems	Cat# 07958935001
Commercial assay or kit	PreCR Repair Mix	New England Biolabs	Cat# M0309L
Commercial assay or kit	SMRTbell Template Prep Kit 1.0-SPv3	Pacific Biosciences	Cat# 100-991-900
Other	GRCz11	NCBI RefSeq	RefSeq:GCF_000002035.6	Zebrafish reference genome
Other	M220 miniTUBE, Red	Covaris	Cat# 4482266	Used to shear DNA on Covaris ultrasonicator
Other	DB MyOne Streptavidin C1	Thermo Fisher Scientific	Cat# 65001	Used to retrieve bait-bound DNA fragments
Other	AMPure XP	Beckman Coulter	Cat# A63881	Size selection beads
Other	Ampure PB	Pacific Biosciences	Cat# 100-265-900	PacBio-compatible size selection beads
Software, algorithm	lima	Pacific Biosciences	lima:v1.0.0; lima:v1.8.0; lima:v1.9.0; lima:v1.11.0
Software, algorithm	ccs	Pacific Biosciences	ccs:v4.2.0
Software, algorithm	pbmarkdup	Pacific Biosciences	pbmarkdup:v1.0.0
Software, algorithm	pbmm2	Pacific Biosciences	pbmm2:v1.3.0
Software, algorithm	samtools	https://doi.org/10.1093/bioinformatics/btp352	samtools:v1.7
Software, algorithm	EMBOSS	https://doi.org/10.1016/s0168-9525(00)02024-2	EMBOSS:v6.6.0.0
Software, algorithm	HMMER	https://doi.org/10.1093/bioinformatics/btt403	HMMER:v3.2.1
Software, algorithm	blastn	https://doi.org/10.1186/1471-2105-10-421	blastn:v2.11.0+
Software, algorithm	hifiasm	https://doi.org/10.1038/s41592-020-01056-5	hifiasm:v0.15.4-r347
Software, algorithm	get_homologues	https://doi.org/10.1128/AEM.02411-13	get_homologues:x86_64–20220516
Software, algorithm	deepvariant	https://doi.org/10.1038/nbt.4235	deepvariant:r1.0
Software, algorithm	GLnexus	https://doi.org/10.1101/343970	Glnexus:v1.2.7–0-g0e74fc4
Software, algorithm	vcftools	https://doi.org/10.1093/bioinformatics/btr330	vcftools:v0.1.16

Appendix 1—table 1

Sequencing scheme for the zebrafish samples.

Libraries sequenced after the introduction of an improved (long run) sequencing chemistry are marked with LR. Samples that yielded no data after sequencing are marked with asterisks.

Individuals	Library	Sequencer
TU01, TU02, TU03, TU06	TU L1	Sequel
TU08, TU10, TU12, TU14	TU L2	Sequel
CGN1, CGN2, CGN3, CGN4	CGN L1	Sequel
CGN5, CGN6, CGN7, CGN8	CGN L2	Sequel
DP07, DP09, DP10, DP12	DP L1	Sequel
DP15, DP20, DP23, DP24, DP25, DP28, DP31, DP34	DP L2	Sequel (LR)
DP03, DP05, DP13, DP16, DP21, DP29, DP31, DP33	DP L3	Sequel (LR)
KG35, KG41, KG42, KG43	KG L1	Sequel
KG03, KG05, KG07, KG12, KG14, KG15, KG18, KG19	KG L2	Sequel (LR)
KG20, KG22, KG24, KG26, KG29, KG32, KG33, KG44	KG L3	Sequel (LR)
SN21, SN23, (SN24*), SN26	SN L1	Sequel
SN03, SN04, SN08, SN09, SN10, SN11, SN12, SN24	SN L2	Sequel II (LR)
SN13, SN14, SN15, SN16, SN17, SN18, SN19, SN20	SN L3	Sequel II (LR)
CHT19, CHT23, CHT26, CHT28	CHT L1	Sequel
CHT01 - CHT07, (CHT13*)	CHT L2	Sequel II (LR)
CHT08, CHT10 - CHT12, CHT14 - CHT16, (SN25*)	CHT L3	Sequel II (LR)

Appendix 1—table 2

PCR program used for barcoding.

For library amplification, the same program was used with 26 or 31 cycles.

Step	Temperature (°C)	Duration
Initialization	98	4 min
Denaturation	98	30 s	{x 12}
Annealing	65	30 s
Elongation	72	12 min
Final elongation	72	20 min
Storage	4	∞

Appendix 1—table 3

qPCR program for the evaluation of enrichment efficiency.

Step	Temperature (°C)	Duration
Initialization	95	12 min
Denaturation	95	15 s	{x 40}
Annealing	65	20 s
Elongation	72	20 s

Appendix 1—table 4

Sequences of qPCR primers used for evaluation of target enrichment.

Gene	Direction	Sequence
il1	+	5’-tgg-tga-acg-tca-tca-tcg-cc-3’
il1	-	5’-tcc-agc-acc-tct-ttt-tct-cca-a-3’
foxo6 intron	+	5’-agt-tct-gtg-tgg-gaa-cag-gg-3’
foxo6 intron	-	5’-gtg-cat-ctt-tag-cgt-tgg-ct-3’
NLR group 1	+	5’-cct-gac-aca-ggt-caa-caa-aac-a-3’
NLR group 1	-	5’-gat-tgt-ctt-ttc-ctt-cag-ccc-ag-3’
NLR group 2	+	5’-tgg-att-ggg-ctg-aag-gga-aa-3’
NLR group 2	-	5’-agg-ttc-agt-cct-tta-gtc-tct-gg-3’
NLR group 3	+	5’-ctg-ctg-gag-gtg-aaa-gat-cag-ac-3’
NLR group 3	-	5’-gat-tgt-tga-gca-gtg-agc-agg-a-3’
NLR group 4	+	5’-tac-ctg-gac-aag-aca-aag-cca-3’
NLR group 4	-	5’-ctc-ctt-ctc-ttc-agc-cca-gtc-3’