DOI: 10.5061/dryad.ncjsxkt3x
title: Amplicon sequencing data for genetic variants at the ASIP locus that determine grey and black fur of the brushtail possum

description: 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&gt;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.
description: 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.
description: # Amplicon sequencing data Access this dataset on Dryad
 [https://doi.org/10.5061/dryad.ncjsxkt3x](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&gt;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](https://www.ncbi.nlm.nih.gov/datasets/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).