作用：在 EDTA 屏蔽后的 Gifu 基因组上用蛋白、Iso-Seq 与 RNA-seq 证据跑 BRAKER3，再按最小长度、N 端甲硫氨酸、无内部终止密码子过滤蛋白及其对应的 CDS 与 GFF3。
入口：Gifu_braker.sh（BRAKER3）；makeblastdb.sh、filter.sh、bedtools.sh、filter_gff_overlaps.py 建库、过滤与去冗余。
输入：屏蔽基因组 ``{PROJ_LOTUS_ZC}/Gifu/09.annotation/03.edta/Gifu_v1.0.fasta.mod.MAKER.masked（来自 01_repeat_edta）；蛋白证据 .../06.braker/douke_pep.fasta；Iso-Seq BAM .../05.isoseq/Gifu_isoforms.sorted.bam；RNA-seq BAM 在 .../04.rnasq/01.align/（逗号分隔的 --bam 列表在 Gifu_braker.sh 第 11 行）；filter.sh 另读 braker.aa、braker.codingseq、braker.gff3，bedtools.sh 读 gene_filter.bed。
输出：.../06.braker 内的 braker.aa、braker.codingseq、braker.gff3；Gifu_filtered.pep.fa、Gifu_filtered.cds.fa、Gifu_filtered.gff3（02_rnaseq_align 用的是 Gifu_filtered.gtf，需自行转换）；overlaps.tsv；filter_gff_overlaps.py 写其第二个位置参数。
运行：bash makeblastdb.sh → bash Gifu_braker.sh → bash filter.sh → python3 filter_gff_overlaps.py <in.gff3> <out.gff3>；filter.sh 调 python AnnotationFilter.py -l 20 -i braker.aa -o Gifu_filtered.pep.fa -c braker.codingseq -C Gifu_filtered.cds.fa -g braker.gff3 -G Gifu_filtered.gff3；bedtools.sh 跑 bedtools intersect -a gene_filter.bed -b gene_filter.bed -wa -wb | awk -F'\t' '$4 < $9' > overlaps.tsv。
工具：BRAKER3 braker.pl --species=Gifu_1 --threads 88 --gff3（singularity exec braker3_sandbox，镜像是本地镜像名而非路径）；AUGUSTUS --AUGUSTUS_CONFIG_PATH={SOFTWARE_ZC}/Augustus-master/config/；BLAST+ makeblastdb -in douke_pep.fasta -dbtype prot -out douke_pep.fasta；bedtools intersect -wa -wb；AnnotationFilter.py -l 20；filter_gff_overlaps.py --strand-aware。
English
**Purpose**: Run BRAKER3 on the EDTA-masked Gifu genome with protein, Iso-Seq and RNA-seq evidence, then filter the predicted proteins (minimum length, N-terminal methionine, no internal stop codon) together with their matching CDS and GFF3 features.
**Entry point**: `Gifu_braker.sh` (BRAKER3); `makeblastdb.sh`, `filter.sh`, `bedtools.sh` and `filter_gff_overlaps.py` build the BLAST database, filter and reduce redundancy.
**Inputs**: masked genome ``{PROJ_LOTUS_ZC}`/Gifu/09.annotation/03.edta/Gifu_v1.0.fasta.mod.MAKER.masked` (from `01_repeat_edta`); protein evidence `.../06.braker/douke_pep.fasta`; Iso-Seq BAM `.../05.isoseq/Gifu_isoforms.sorted.bam`; RNA-seq BAMs in `.../04.rnasq/01.align/` (the comma-separated `--bam` list is on line 11 of `Gifu_braker.sh`); `filter.sh` also reads `braker.aa`, `braker.codingseq`, `braker.gff3`, and `bedtools.sh` reads `gene_filter.bed`.
**Outputs**: `braker.aa`, `braker.codingseq`, `braker.gff3` in `.../06.braker`; `Gifu_filtered.pep.fa`, `Gifu_filtered.cds.fa`, `Gifu_filtered.gff3` (note that `02_rnaseq_align` uses `Gifu_filtered.gtf`); `overlaps.tsv`; `filter_gff_overlaps.py` writes its second positional argument.
**Run**: `bash makeblastdb.sh` → `bash Gifu_braker.sh` → `bash filter.sh` → `python3 filter_gff_overlaps.py `; `filter.sh` calls `python AnnotationFilter.py -l 20 -i braker.aa -o Gifu_filtered.pep.fa -c braker.codingseq -C Gifu_filtered.cds.fa -g braker.gff3 -G Gifu_filtered.gff3`; `bedtools.sh` runs `bedtools intersect -a gene_filter.bed -b gene_filter.bed -wa -wb | awk -F'\t' '$4 < $9' > overlaps.tsv`.
**Tools**: BRAKER3 `braker.pl --species=Gifu_1 --threads 88 --gff3` (via `singularity exec braker3_sandbox`, a local image name rather than a path); AUGUSTUS `--AUGUSTUS_CONFIG_PATH=`{SOFTWARE_ZC}`/Augustus-master/config/`; BLAST+ `makeblastdb -in douke_pep.fasta -dbtype prot -out douke_pep.fasta`; bedtools `intersect -wa -wb`; `AnnotationFilter.py -l 20`; `filter_gff_overlaps.py --strand-aware`.