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Amplicon sequencing data for genetic variants at the ASIP locus that determine grey and black fur of the brushtail possum

Cite this dataset

Bond, Donna (2024). Amplicon sequencing data for genetic variants at the ASIP locus that determine grey and black fur of the brushtail possum [Dataset]. Dryad. https://doi.org/10.5061/dryad.ncjsxkt3x

Abstract

The possession of fur or hair is a defining characteristic of mammals and can occur in a variety of colours and patterns. While genetic determinants of coat colour are well described in eutherian ‘placental’ mammals, the other major mammalian infraclass, marsupials, is grossly understudied. The fur of the common brushtail possum (Trichosurus vulpecula), an iconic native mammal found throughout Australia and introduced into Aotearoa New Zealand, possesses two main colour morphs: grey and black. To identify genetic variants associated with coat colour, we performed a genome wide association study (GWAS) with genotype by sequencing (GBS) data. Single nucleotide polymorphisms (SNPs) on chromosome 3, close to the agouti signalling protein (ASIP) gene that controls the temporal and spatial distribution of pigments in eutherian mammals, were identified. Fine-mapping identified a C>T variant at chr3:100,483,705 that results in a p.Arg115Cys substitution of the ASIP protein, and animals homozygous for this variant have black fur. In addition to uncovering the first genetic determinant of coat colour in a natural marsupial population, comparative analysis of ASIP in divergent marsupial species identified the Dasyurids as having accelerated evolution, reflecting their well described diversity of coat colour and pattern.

README: Amplicon sequencing data

Access this dataset on Dryad https://doi.org/10.5061/dryad.ncjsxkt3x

The dataset is a list of fastq sequencing files, with the raw (unprocessed) amplicon sequencing reads for each sample. The amplicon was generated by a dual-indexing, four-primer PCR-based assay, and covers the genetic variant identified in this study that resides within the ASIP gene (chr3:100,483,705 (c.343C>T)). The amplicon was sequenced on the Illumina iSeq100 and 150bp paired-end sequencing reads were generated. This data is for samples presented in Figure 3 - samples from the Lawrence and Dunedin populations.

Description of the data and file structure

For each sample, there are 2 raw sequencing data files - R1 and R2 - that form the paired-end reads and need to be processed together. See the Supplementary S2 file for a summary of the processed data used to generate Figure 3, and additional metadata information.

Population Sample Name Raw sequencing data file - R1 Raw sequencing data file - R2
Lawrence Grey 1 ASIP-17_S17_L001_R1_001.fastq.gz ASIP-17_S17_L001_R2_001.fastq.gz
Lawrence Grey 2 ASIP-18_S18_L001_R1_001.fastq.gz ASIP-18_S18_L001_R2_001.fastq.gz
Lawrence Grey 3 ASIP-19_S19_L001_R1_001.fastq.gz ASIP-19_S19_L001_R2_001.fastq.gz
Lawrence Grey 4 ASIP-20_S20_L001_R1_001.fastq.gz ASIP-20_S20_L001_R2_001.fastq.gz
Lawrence Grey 5 ASIP-21_S21_L001_R1_001.fastq.gz ASIP-21_S21_L001_R2_001.fastq.gz
Lawrence Grey 6 ASIP-22_S22_L001_R1_001.fastq.gz ASIP-22_S22_L001_R2_001.fastq.gz
Lawrence Grey 7 ASIP-23_S23_L001_R1_001.fastq.gz ASIP-23_S23_L001_R2_001.fastq.gz
Lawrence Grey 8 ASIP-24_S24_L001_R1_001.fastq.gz ASIP-24_S24_L001_R2_001.fastq.gz
Lawrence Grey 9 ASIP-25_S25_L001_R1_001.fastq.gz ASIP-25_S25_L001_R2_001.fastq.gz
Lawrence Grey 10 ASIP-26_S26_L001_R1_001.fastq.gz ASIP-26_S26_L001_R2_001.fastq.gz
Lawrence Grey 11 ASIP-27_S27_L001_R1_001.fastq.gz ASIP-27_S27_L001_R2_001.fastq.gz
Lawrence Grey 12 ASIP-28_S28_L001_R1_001.fastq.gz ASIP-28_S28_L001_R2_001.fastq.gz
Lawrence Grey 13 ASIP-29_S29_L001_R1_001.fastq.gz ASIP-29_S29_L001_R2_001.fastq.gz
Lawrence Grey 14 ASIP-30_S30_L001_R1_001.fastq.gz ASIP-30_S30_L001_R2_001.fastq.gz
Lawrence Grey 15 ASIP-31_S31_L001_R1_001.fastq.gz ASIP-31_S31_L001_R2_001.fastq.gz
Lawrence Grey 16 ASIP-32_S32_L001_R1_001.fastq.gz ASIP-32_S32_L001_R2_001.fastq.gz
Lawrence Grey 17 ASIP-33_S33_L001_R1_001.fastq.gz ASIP-33_S33_L001_R2_001.fastq.gz
Lawrence Grey 18 ASIP-34_S34_L001_R1_001.fastq.gz ASIP-34_S34_L001_R2_001.fastq.gz
Lawrence Grey 19 ASIP-35_S35_L001_R1_001.fastq.gz ASIP-35_S35_L001_R2_001.fastq.gz
Lawrence Grey 20 ASIP-36_S36_L001_R1_001.fastq.gz ASIP-36_S36_L001_R2_001.fastq.gz
Lawrence Grey 21 ASIPG21-8fold_S54_L001_R1_001.fastq.gz ASIPG21-8fold_S54_L001_R2_001.fastq.gz
Lawrence Grey 22 ASIP-38_S38_L001_R1_001.fastq.gz ASIP-38_S38_L001_R2_001.fastq.gz
Lawrence Grey 23 ASIP-39_S39_L001_R1_001.fastq.gz ASIP-39_S39_L001_R2_001.fastq.gz
Lawrence Grey 24 ASIP-40_S40_L001_R1_001.fastq.gz ASIP-40_S40_L001_R2_001.fastq.gz
Lawrence Grey 25 ASIP-41_S41_L001_R1_001.fastq.gz ASIP-41_S41_L001_R2_001.fastq.gz
Lawrence Grey 26 ASIP-42_S42_L001_R1_001.fastq.gz ASIP-42_S42_L001_R2_001.fastq.gz
Lawrence Grey 27 ASIP-43_S43_L001_R1_001.fastq.gz ASIP-43_S43_L001_R2_001.fastq.gz
Lawrence Grey 28 ASIP-44_S44_L001_R1_001.fastq.gz ASIP-44_S44_L001_R2_001.fastq.gz
Lawrence Black 1 ASIP-49_S49_L001_R1_001.fastq.gz ASIP-49_S49_L001_R2_001.fastq.gz
Lawrence Black 2 ASIP-50_S50_L001_R1_001.fastq.gz ASIP-50_S50_L001_R2_001.fastq.gz
Lawrence Black 3 ASIP-51_S51_L001_R1_001.fastq.gz ASIP-51_S51_L001_R2_001.fastq.gz
Lawrence Black 4 ASIP-52_S52_L001_R1_001.fastq.gz ASIP-52_S52_L001_R2_001.fastq.gz
Lawrence Black 5 ASIP-53_S53_L001_R1_001.fastq.gz ASIP-53_S53_L001_R2_001.fastq.gz
Lawrence Black 6 ASIP-54_S54_L001_R1_001.fastq.gz ASIP-54_S54_L001_R2_001.fastq.gz
Lawrence Black 7 ASIP-55_S55_L001_R1_001.fastq.gz ASIP-55_S55_L001_R2_001.fastq.gz
Lawrence Black 8 ASIP-56_S56_L001_R1_001.fastq.gz ASIP-56_S56_L001_R2_001.fastq.gz
Lawrence Black 9 ASIP-57_S57_L001_R1_001.fastq.gz ASIP-57_S57_L001_R2_001.fastq.gz
Lawrence Black 10 ASIP-58_S58_L001_R1_001.fastq.gz ASIP-58_S58_L001_R2_001.fastq.gz
Lawrence Black 11 ASIP-59_S59_L001_R1_001.fastq.gz ASIP-59_S59_L001_R2_001.fastq.gz
Lawrence Black 12 ASIP-60_S60_L001_R1_001.fastq.gz ASIP-60_S60_L001_R2_001.fastq.gz
Lawrence Black 13 ASIP-61_S61_L001_R1_001.fastq.gz ASIP-61_S61_L001_R2_001.fastq.gz
Lawrence Black 14 ASIP-62_S62_L001_R1_001.fastq.gz ASIP-62_S62_L001_R2_001.fastq.gz
Lawrence Black 15 ASIP-63_S63_L001_R1_001.fastq.gz ASIP-63_S63_L001_R2_001.fastq.gz
Lawrence Black 16 ASIP-64_S64_L001_R1_001.fastq.gz ASIP-64_S64_L001_R2_001.fastq.gz
Lawrence Black 17 ASIP-65_S65_L001_R1_001.fastq.gz ASIP-65_S65_L001_R2_001.fastq.gz
Lawrence Black 18 ASIP-66_S66_L001_R1_001.fastq.gz ASIP-66_S66_L001_R2_001.fastq.gz
Lawrence Black 19 ASIP-67_S67_L001_R1_001.fastq.gz ASIP-67_S67_L001_R2_001.fastq.gz
Dunedin Puku iSeq061-Otago-ASIPshort-01_S1_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-01_S1_L001_R2_001.fastq.gz
Dunedin Sandy iSeq061-Otago-ASIPshort-02_S2_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-02_S2_L001_R2_001.fastq.gz
Dunedin Sheila iSeq061-Otago-ASIPshort-03_S3_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-03_S3_L001_R2_001.fastq.gz
Dunedin 190320_FG_WSG2 ID4-ASIPshort_S55_L001_R1_001.fastq.gz ID4-ASIPshort_S55_L001_R2_001.fastq.gz
Dunedin 030620_FG_WSG1 ID6-ASIPshort_S46_L001_R1_001.fastq.gz ID6-ASIPshort_S46_L001_R2_001.fastq.gz
Dunedin 030720_FG_SDR1 ID8-ASIPshort_S56_L001_R1_001.fastq.gz ID8-ASIPshort_S56_L001_R2_001.fastq.gz
Dunedin 140720_FG_BB1 iSeq061-Otago-ASIPshort-04_S4_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-04_S4_L001_R2_001.fastq.gz
Dunedin 150720_FR_BB1 iSeq061-Otago-ASIPshort-05_S5_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-05_S5_L001_R2_001.fastq.gz
Dunedin 280720_FG_SDR1 ID14-ASIPshort_S57_L001_R1_001.fastq.gz ID14-ASIPshort_S57_L001_R2_001.fastq.gz
Dunedin 040820_FG_BB1 ID16-ASIPshort_S58_L001_R1_001.fastq.gz ID16-ASIPshort_S58_L001_R2_001.fastq.gz
Dunedin 120820_FG_WSG2 iSeq061-Otago-ASIPshort-30_S30_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-30_S30_L001_R2_001.fastq.gz
Dunedin 180820_FB_SDR1 ID20-ASIPshort_S59_L001_R1_001.fastq.gz ID20-ASIPshort_S59_L001_R2_001.fastq.gz
Dunedin 160620_MG_SDR1 P2-ASIPshort_S47_L001_R1_001.fastq.gz P2-ASIPshort_S47_L001_R2_001.fastq.gz
Dunedin 130320_MB_SDR1 P3-ASIPshort_S60_L001_R1_001.fastq.gz P3-ASIPshort_S60_L001_R2_001.fastq.gz
Dunedin 170320_FR_SDR1 iSeq061-Otago-ASIPshort-06_S6_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-06_S6_L001_R2_001.fastq.gz
Dunedin 190320_MR_WSG1 P5-ASIPshort_S61_L001_R1_001.fastq.gz P5-ASIPshort_S61_L001_R2_001.fastq.gz
Dunedin 250620_PY01 iSeq061-Otago-ASIPshort-07_S7_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-07_S7_L001_R2_001.fastq.gz
Dunedin 120320_MB_SDR2 iSeq061-Otago-ASIPshort-08_S8_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-08_S8_L001_R2_001.fastq.gz
Dunedin 150720_MG_BB2 P9-ASIPshort_S48_L001_R1_001.fastq.gz P9-ASIPshort_S48_L001_R2_001.fastq.gz
Dunedin 250620_FG_WSG1 iSeq061-Otago-ASIPshort-09_S9_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-09_S9_L001_R2_001.fastq.gz
Dunedin 100620_MG_BB1 P11-ASIPshort_S49_L001_R1_001.fastq.gz P11-ASIPshort_S49_L001_R2_001.fastq.gz
Dunedin 120320_MR_SDR1 iSeq061-Otago-ASIPshort-10_S10_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-10_S10_L001_R2_001.fastq.gz
Dunedin 180320_MG_WSG1 iSeq061-Otago-ASIPshort-11_S11_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-11_S11_L001_R2_001.fastq.gz
Dunedin 100720_MG_SDR2 iSeq061-Otago-ASIPshort-12_S12_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-12_S12_L001_R2_001.fastq.gz
Dunedin 210720_MG_WSG1 iSeq061-Otago-ASIPshort-13_S13_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-13_S13_L001_R2_001.fastq.gz
Dunedin 110820_FG_WSG1 P17-ASIPshort_S50_L001_R1_001.fastq.gz P17-ASIPshort_S50_L001_R2_001.fastq.gz
Dunedin 120820_MG_WSG1 iSeq061-Otago-ASIPshort-14_S14_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-14_S14_L001_R2_001.fastq.gz
Dunedin 130820_MG_WSG1 iSeq061-Otago-ASIPshort-15_S15_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-15_S15_L001_R2_001.fastq.gz
Dunedin 190820_MB_SDR1 iSeq061-Otago-ASIPshort-16_S16_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-16_S16_L001_R2_001.fastq.gz
Dunedin 030920_MB_SDR1 iSeq061-Otago-ASIPshort-17_S17_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-17_S17_L001_R2_001.fastq.gz
Dunedin 170920_FB_SDR1 iSeq061-Otago-ASIPshort-18_S18_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-18_S18_L001_R2_001.fastq.gz
Dunedin 090421_MB_SDR1 iSeq061-Otago-ASIPshort-19_S19_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-19_S19_L001_R2_001.fastq.gz
Dunedin 140421_MG_WSG1 iSeq061-Otago-ASIPshort-20_S20_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-20_S20_L001_R2_001.fastq.gz
Dunedin 200421_MB_SDR1 iSeq061-Otago-ASIPshort-21_S21_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-21_S21_L001_R2_001.fastq.gz
Dunedin 040521_MB_SDR1 iSeq061-Otago-ASIPshort-22_S22_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-22_S22_L001_R2_001.fastq.gz
Dunedin 180521_MG_PF3 P38-ASIPshort_S51_L001_R1_001.fastq.gz P38-ASIPshort_S51_L001_R2_001.fastq.gz
Dunedin 180521_MG_PF1 P39-ASIPshort_S52_L001_R1_001.fastq.gz P39-ASIPshort_S52_L001_R2_001.fastq.gz
Dunedin 180521_FG_PF2 P40-ASIPshort_S53_L001_R1_001.fastq.gz P40-ASIPshort_S53_L001_R2_001.fastq.gz
Dunedin 200521_MR_PF2 iSeq061-Otago-ASIPshort-26_S26_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-26_S26_L001_R2_001.fastq.gz
Dunedin 250521_MG_PF1 P42-ASIPshort_S54_L001_R1_001.fastq.gz P42-ASIPshort_S54_L001_R2_001.fastq.gz
Dunedin 030621_MR_PF1 iSeq061-Otago-ASIPshort-28_S28_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-28_S28_L001_R2_001.fastq.gz
Dunedin 090621_MG_PF1 iSeq061-Otago-ASIPshort-29_S29_L001_R1_001.fastq.gz iSeq061-Otago-ASIPshort-29_S29_L001_R2_001.fastq.gz

Code/Software

The raw data was processed to generate Figure 3 (processed data is available in Supplementary File 2). The processing involved importing the sequencing reads into Geneious v2022.2.2 and trimming the reads (removing adapters) using the BBDuk plugin. Trimmed reads were aligned to an ‘in silico’ PCR product, which corresponds to the sequence extracted from the possum reference genome (GCA_011100635.1: chr3:100,483,628-100,483,799; see Supplementary Table 2 for corresponding primer sequences). The ‘Find Variations/SNPs’ feature within Geneious was used to determine the nucleotide sequence(s) at the SNP site of interest, with a minimum variant frequency of 0.2. The coverage (total number of reads), reference read count and variant read count was extracted for each amplicon (Supplementary File 2) and used to plot the variant frequency for each sample (Figure 3).

Methods

A dual-indexing, four-primer PCR-based assay was used for amplicon sequencing, as described in Bond et al., 2023. Both the first and second round of PCR amplification were performed using Phusion High-Fidelity DNA Polymerase (M0530L; New England Biolabs) according to the manufacturers’ protocol. For the first round of amplification, approximately 10–25 ng of DNA was used in the following PCR mix: 1x HF Buffer with 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 µM of each primer, and 0.02 U/µL units of Phusion, with the following PCR cycling conditions:  98 °C for 3 min; 27 cycles of 98 °C for 10 s, 62 °C for 20 s, and 72 °C for 15 s; 72 °C for 2 mins. The PCR reaction was cleaned up and sized selected using 0.9X SPRI beads diluted in standard PEG buffer (18% w/v polyethylene glycol 8000 (PEG), 1 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 0.05% v/v Tween-20) (Oberacker et al., 2023). The DNA was eluted in 10 µL nuclease-free water and 2 µL was used as a template in a second round of PCR amplification (using the same PCR conditions as described above) with 0.2 µM of each second step primer (indexed Truseq-type oligos for Illumina sequencing). PCR cycling parameters for the second step PCR were: 98 °C for 3 min; 5 cycles of 98 °C for 10 s, 62 °C for 20 s, and 72 °C for 15 s; 72 °C for 2 min. The final libraries were pooled in equimolar amounts, cleaned-up and size-selected using 0.9X SPRI beads diluted in standard PEG buffer and sequenced on the iSeq100 (Illumina) to generate 150 bp paired-end reads. All primers used for amplicon sequencing are given in S2 Table.

Funding

Ministry of Business, Innovation and Employment, Award: UOOX1909

Predator Free 2050 Ltd, Award: SS2/01/01

Predator Free 2050 Ltd, Award: Jobs for Nature

University of Otago