玉米根尖热处理单细胞数据分析
玉米根尖热处理单细胞数据分析
1.参考基因组下载
wget -c https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-62/fasta/zea_mays/dna/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.dna.toplevel.fa.gz
wget -c https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-62/gff3/zea_mays/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.62.gff3.gz
wget -c https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-62/gtf/zea_mays/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.62.gtf.gz
2. cellranger 索引建立
#可选操作，选择蛋白编码的基因：
#cellranger mkgtf P_aphrodite_genomic_scaffold_v1.0_gene.gtf P_aphrodite_genomic_scaffold_v1.0_gene.filtered.gtf --attribute=gene_biotype:protein_coding
#10X官方参考基因组索引建立参考：https://www.10xgenomics.com/cn/support/software/space-ranger/latest/advanced/custom-references
cellranger mkref --nthreads 20 --genome=B73 --fasta=Zea_mays.Zm-B73-REFERENCE-NAM-5.0.dna.toplevel.fa --genes=Zea_mays.Zm-B73-REFERENCE-NAM-5.0.62.gtf
3.示例数据下载
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070725/CRR1070725_f1.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070726/CRR1070726_f1.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070727/CRR1070727_f1.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070728/CRR1070728_f1.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070725/CRR1070725_r2.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070726/CRR1070726_r2.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070727/CRR1070727_r2.fq.gz
wget -c ftp://download.big.ac.cn/gsa2/CRA015077/CRR1070728/CRR1070728_r2.fq.gz
4. 改名字 方便cellranger读入
5. 准备样本信息文件 meta.tsv ：
SampleID Accession Group
PU1 CRR1070725 CK
PU2 CRR1070726 CK
EW01 CRR1070727 HS
EW02 CRR1070728 HS
6. cellranger 分析
cat ../meta.tsv|sed '1d'|while read SampleIDAccessionGroup;do
echo "Processing $SampleID"
cellranger count --id=$SampleID \
--fastqs=$datadir/ \
--sample=$SampleID \
--localcores 20 --create-bam=true \
--transcriptome=$refdir
#velocyto run10x -m $refdir/genes/mm39_rmsk.gtf ../01.cellranger/$SampleID/ $refdir/genes/genes.gtf
done
参考文献：
Wang, T., Wang, F., Deng, S. et al. Single-cell transcriptomes reveal spatiotemporal heat stress response in maize roots. Nat Commun 16, 177 (2025). https://doi.org/10.1038/s41467-024-55485-3
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