{"id":667,"date":"2025-12-26T15:11:55","date_gmt":"2025-12-26T07:11:55","guid":{"rendered":"https:\/\/www.kz-hub.tech\/?p=667"},"modified":"2025-12-26T15:13:44","modified_gmt":"2025-12-26T07:13:44","slug":"cellranger-mkref","status":"publish","type":"post","link":"https:\/\/www.kz-hub.tech\/index.php\/2025\/12\/26\/cellranger-mkref\/","title":{"rendered":"cellranger mkref \u6784\u5efa\u53c2\u8003\u57fa\u56e0\u6587\u4ef6"},"content":{"rendered":"

\u5b98\u65b9\u6559\u7a0b\uff1ahttps:\/\/www.10xgenomics.com\/support\/software\/cell-ranger\/latest\/tutorials\/cr-tutorial-mr<\/a><\/p>\n

create_count_ref_Ensembl_Gencode.sh<\/p>\n

# Genome metadata\ngenome="GRCh38_Ensembl_GENCODE48"\nversion="Ensembl114"\n\n# Set up source and build directories\nbuild="GRCh38_Ensembl_GENCODE48_build"\nmkdir -p "$build"\n\nsource="\/data02\/zhangmengmeng\/database\/hg38"\nfasta_in="${source}\/Homo_sapiens.GRCh38.114.dna.primary_assembly.fa"\n#gtf_in="${source}\/Homo_sapiens.GRCh38.114.gtf"\ngtf_in="${source}\/gencode.v48.primary_assembly.basic.annotation.gtf"\n\n# Modify sequence headers in the Ensembl FASTA to match the file\n# "GRCh38.primary_assembly.genome.fa" from GENCODE. Unplaced and unlocalized\n# sequences such as "KI270728.1" have the same names in both versions.\n#\n# Input FASTA:\n#   >1 dna:chromosome chromosome:GRCh38:1:1:248956422:1 REF\n#\n# Output FASTA:\n#   >chr1 1\nfasta_modified="$build\/$(basename "$fasta_in").modified"\n# sed commands:\n# 1. Replace metadata after space with original contig name, as in GENCODE\n# 2. Add "chr" to names of autosomes and sex chromosomes\n# 3. Handle the mitochrondrial chromosome\ncat "$fasta_in" \\\n    | sed -E 's\/^>(\\S+).*\/>\\1 \\1\/' \\\n    | sed -E 's\/^>([0-9]+|[XY]) \/>chr\\1 \/' \\\n    | sed -E 's\/^>MT \/>chrM \/' \\\n    > "$fasta_modified"\n\n# Remove version suffix from transcript, gene, and exon IDs in order to match\n# previous Cell Ranger reference packages\n#\n# Input GTF:\n#     ... gene_id "ENSG00000223972.5"; ...\n# Output GTF:\n#     ... gene_id "ENSG00000223972"; gene_version "5"; ...\ngtf_modified="$build\/$(basename "$gtf_in").modified"\n# Pattern matches Ensembl gene, transcript, and exon IDs for human or mouse:\nID="(ENS(MUS)?[GTE][0-9]+)\\.([0-9]+)"\ncat "$gtf_in" \\\n    | sed -E 's\/gene_id "'"$ID"'";\/gene_id "\\1"; gene_version "\\3";\/' \\\n    | sed -E 's\/transcript_id "'"$ID"'";\/transcript_id "\\1"; transcript_version "\\3";\/' \\\n    | sed -E 's\/exon_id "'"$ID"'";\/exon_id "\\1"; exon_version "\\3";\/' \\\n    > "$gtf_modified"\n\n# Define string patterns for GTF tags\n# NOTES:\n# Since Ensembl 110, polymorphic pseudogenes are now just protein_coding.\n# Readthrough genes are annotated with the readthrough_transcript tag.\nBIOTYPE_PATTERN=\\\n"(protein_coding|protein_coding_LoF|lncRNA|\\\nIG_C_gene|IG_D_gene|IG_J_gene|IG_LV_gene|IG_V_gene|\\\nIG_V_pseudogene|IG_J_pseudogene|IG_C_pseudogene|\\\nTR_C_gene|TR_D_gene|TR_J_gene|TR_V_gene|\\\nTR_V_pseudogene|TR_J_pseudogene)"\nGENE_PATTERN="gene_type \\"${BIOTYPE_PATTERN}\\""\nTX_PATTERN="transcript_type \\"${BIOTYPE_PATTERN}\\""\nREADTHROUGH_PATTERN="tag \\"readthrough_transcript\\""\n\n# Construct the gene ID allowlist. We filter the list of all transcripts\n# based on these criteria:\n#   - allowable gene_type (biotype)\n#   - allowable transcript_type (biotype)\n#   - no "readthrough_transcript" tag\n# We then collect the list of gene IDs that have at least one associated\n# transcript passing the filters.\ncat "$gtf_modified" \\\n    | awk '$3 == "transcript"' \\\n    | grep -E "$GENE_PATTERN" \\\n    | grep -E "$TX_PATTERN" \\\n    | grep -Ev "$READTHROUGH_PATTERN" \\\n    | sed -E 's\/.*(gene_id "[^"]+").*\/\\1\/' \\\n    | sort \\\n    | uniq \\\n    > "${build}\/gene_allowlist"\n\n# NOTES:\n# Since Ensembl 110, the PAR locus genes are included on chrY as copies of chrX\n# Using the GRCh38.p13 assembly hard masks these regions on chrY, but removing the\n# chrY PAR genes is still desirable so they do not end up as extra entries in the output.\n# The awk command below excludes all PAR_Y genes, including XGY2.\n# The non-coding gene XGY2 straddles the PAR1 boundary on chrY, and is homologous to XG on chrX.\n# GRCh38-2024-A excludes XGY2, but includes SRY and ENSG00000286130, which are in an intron of XGY2,\n# and RPS4Y1, which overlaps XGY2.\n\n# Filter the GTF file based on the gene allowlist\ngtf_filtered="${build}\/$(basename "$gtf_in").filtered"\n# Copy header lines beginning with "#"\ngrep -E "^#" "$gtf_modified" > "$gtf_filtered"\n# Filter to the gene allowlist, and then remove PAR_Y genes\ngrep -Ff "${build}\/gene_allowlist" "$gtf_modified" \\\n    | awk -F "\\t" '$1 != "chrY" || $1 == "chrY" && $4 >= 2752083 && $4 < 56887903 && !\/ENSG00000290840\/' \\\n    >> "$gtf_filtered"\n\n# Create reference package\ncellranger mkref --ref-version="$version" \\\n    --genome="$genome" --fasta="$fasta_modified" --genes="$gtf_filtered" \\\n    --nthreads=8<\/code><\/pre>\n","protected":false},"excerpt":{"rendered":"
\u5b98\u65b9\u6559\u7a0b\uff1ahttps:\/\/www.10xgenomics.com\/support\/software\/cell-…<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-667","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"_links":{"self":[{"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/posts\/667","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/comments?post=667"}],"version-history":[{"count":2,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/posts\/667\/revisions"}],"predecessor-version":[{"id":669,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/posts\/667\/revisions\/669"}],"wp:attachment":[{"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/media?parent=667"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/categories?post=667"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.kz-hub.tech\/index.php\/wp-json\/wp\/v2\/tags?post=667"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}