DNA Sequencing and SNP Calling
Three
to five individuals were randomly selected from each population whose
phenotype was measured to extract DNA. Total genomic DNA was extracted
using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle &
Doyle, 1987).
Illumina
Xten from Biomarker Technologies, Inc. (Beijing, China) was used for
genomic library generation and sequencing with 2 × 150 bp paired reads.
Furthermore, the raw reads of Semiaquilegia adoxoide s (SRR437677)
were downloaded from the NCBI SRA database
(http://www.ncbi.nlm.nih.gov/sra) to be used as an outgroup.
To
obtain high-quality genomes, all the reads were assessed using FastQC
(Andrew, 2010) and filtered as follows: reads with adapters and reads
with more than a 10% N content or more than 50% low-quality bases
(quality value of less than 10) were removed. Low-quality reads were
removed using NGStoolkit (Mulcare, 2004). The clean reads of each sample
were mapped to the genome sequence of A. coerulea downloaded from
the previous study of Filiault et al. (Filiault et al., 2018) using BWA
v.0.7.12 with default parameters (H. Li & Durbin, 2009). SAMtools
v.0.1.18 was used to sort reads (H. Li et al., 2009). The
HaplotypeCaller, GenotypeGVCFs and CombineGVCFs modules in
GATK
v.4.1.8.0 were used to produce accurate SNP calls (McKenna et al.,
2010). To improve the quality of SNPs, the VariantFiltration module in
GATK v4.1.8.0 was used for filtration with the following parameters:
–filter-name FilterQual –filter-expression ”QUAL < 30.0”
–filter-name FilterQD –filter-expression ”QD < 2.0”
–filter-name FilterMQ –filter-expression ”MQ < 40.0”
–filter-name FilterFS –filter-expression ”FS > 60.0”
-window 5 -cluster 2. Next, VCFtools v0.1.13 (Danecek et al., 2011) was
used to remove variants that 1) showed a minor allele frequency (MAF) of
0.02 or less, 2) were not balletic variants, 3) showed a sequencing
depth of less than 5, and 4) showed a missing rate exceeding 0.5. The
filtered data were used for subsequent analysis. After filtering, each
SNP was annotated using
SnpEff
v.5.0 (Cingolani et al., 2012).