Genome assembly and SV analysis pipelines for Drosophila pseudoobscura (ST) and Drosophila persimilis (M40) - Extended methods.

This section shows the genome assembly pipeline used (from paper) for the D. pseudoobscura (ST) and D. persimilis (M40). This pipeline implements a hybrid assembly using Illumina short reads and PacBio CLR reads.

Step 1

Generation of a PacBio-only, gap-filled and polished assembly: this assembly was assembled directly by Pacific Biosciences using HGAP-Arrow with default parameters (please refer to the HGAP github page for details). The assembly statistics for each species are available at the Supplementary_Tables.xlsx file (Table S13).

Step 1.1

• Gap-filling of the PacBio-only assemblies using PbJelly.

Commands:

Jelly.py mapping jellyProtocol.xml

Jelly.py support jellyProtocol.xml

Jelly.py extraction jellyProtocol.xml

Jelly.py assembly jellyProtocol.xml -x --nproc=8

NOTE: Please refere to the jellyProtocol.xml for full parameter details.

Step 1.2

• Polishing using Pilon

Commands:

bwa mem -t 5 st_pbjelly.fasta file_1.fastq file_2.fastq > pbjelly.sam 2> stderror.txt

samtools view -bS pbjelly.sam > pbjelly.bam 2> stderror_samtoolsview.txt

samtools sort -@ 5 -o pbjelly_sorted.bam pbjelly.bam 2> stderror_samtoolssort.txt

samtools view -b -F 12 pbjelly_sorted.bam > pbjelly_mapped_sorted.bam 2> stderror_samtoolsmap.txt

samtools index pbjelly_mapped_sorted.bam 2> stderror_samtoolsindex.txt

java -Xmx30G -jar pilon-1.22.jar --genome pbjelly.fasta --frags pbjelly_mapped_sorted.bam --threads 5 --changes 2> stderror_pilon.txt

Step 2.

Generation of a hybrid assembly using CLR reads and Illumina paired-end reads using DBG2OLC.

Commands:

SparseAssembler LD 0 k 51 g 15 NodeCovTh 1 EdgeCovTh 0 GS 171281433 i1 file_1.fastq o1 file_2.fastq > sparseAssembler_test1.log

SparseAssembler LD 1 k 51 g 15 NodeCovTh 2 EdgeCovTh 1 GS 171281433 i1 st_1.fastq o1 st_2.fastq > sparseAssembler_test1.log

DBG2OLC k 17 KmerCovTh 2 MinOverlap 20 AdaptiveTh 0.002 LD1 0 MinLen 200 Contigs Contigs.txt RemoveChimera 1 f pacbio.fasta

Step 3.

Final scaffolding step using quickmerge

Commands:

nucmer -l 100 -p out -t 10 pacbiopolished.fasta hybrid.fasta 2> stderror_nucmer.txt

delta-filter -i 95 -r -q out.delta > out.rq.delta 2> stderror_deltafilter.txt

quickmerge -d out.rq.delta -q hybrid.fasta -r pacbiopolished.fasta -hco 5.0 -c 1.5 -l n -ml m 2> stderror_quickmerge.txt

Structural variation analysis.

This section shows the followed approach for the structural variation analysis that includes INDEL and CNV calling using CLR reads and whole genome alignments.

Step 1.

SV calling using svim. Pipeline is contained in the SVcalling_reciprocalvalidation_CLRreads.sh file.

Step 2.

SV calling using svmu. svmu commands used for all species pair-wise comparisons.

nucmer -t 10 --maxmatch --preferenceix=file.mm reference_genome.fasta query_genome.fasta

lastz reference_genome.fasta[multiple] query_genome.fastaa[multiple] --chain --format=general:name1,strand1,start1,end1,name2,strand2,start2,end2 > file_lastz.txt

svmu file.mm.delta reference_genome.fasta query_genome.fasta 5 l file_lastz.txt file-svmu

Step 3.

SV polarization of D. pseudoobscura and D. persimilis variants with D. miranda. Overall approach and scripts are available at the SV folder.

Syri SV calling.

SV calling using syri for each species pair.

Command:

minimap2 -ax asm5 -t20 --eqx refgenome qrygenome > out.sam

python3 syri -c out.sam -r refgenome -q qrygenome -k -F S

Genome annotation post-processing.

Please refer to the genomeAnnotation_postprocessing.sh for the complete pipeline.