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Snakefile
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Snakefile
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import pathlib
import os
import sys
import gzip
import shutil
import requests
import pandas as pd
from glob import glob
from itertools import product
from snakemake.utils import validate, min_version
##### load config and point to scripts #####
# configfile: "config/config.yml"
if "--configfile" in sys.argv:
i = sys.argv.index("--configfile")
config_path = sys.argv[i + 1]
configfile: config_path
else:
config_path = "config/config.yml"
configfile: config_path
SCRIPT_DIR = srcdir("scripts")
############################
# Validate kit_configs.csv #
############################
kit_fn = config.get("KIT_CONFIGS")
kit_df = pd.read_csv(kit_fn, sep=",", comment="#")
#######################
# Validate config.yml #
#######################
if not config.get("SAMPLE_SHEET"):
raise Exception(f"Please define SAMPLE_SHEET in the {config_path}")
elif not os.path.exists(config["SAMPLE_SHEET"]):
raise Exception(f"Path specified for SAMPLE_SHEET in {config_path} not found!")
sample_sheet = pd.read_csv(
config.get("SAMPLE_SHEET", "./config/samples.csv"), sep=",", comment="#"
).set_index("run_id", drop=True)
RUN_IDS = sample_sheet.index
if not config.get("REF_GENOME_DIR"):
raise Exception(f"Please define REF_GENOME_DIR in the {config_path}")
elif not os.path.exists(config["REF_GENOME_DIR"]):
raise Exception(f"Path specified for REF_GENOME_DIR in {config_path} not found!")
REF_GENOME_DIR = pathlib.Path(config["REF_GENOME_DIR"])
REF_GENOME_FASTA = REF_GENOME_DIR / "fasta/genome.fa"
REF_GENES_GTF = REF_GENOME_DIR / "genes/genes.gtf"
PLOT_GENES = list(map(lambda x: x.upper(), config.get("UMAP_PLOT_GENES", None).split(",")))
BC_LONGLIST_DIR = Path(srcdir("data"))
BC_LONGLIST_3PRIME = BC_LONGLIST_DIR / "3M-february-2018.txt.gz"
BC_LONGLIST_5PRIME = BC_LONGLIST_DIR / "737K-august-2016.txt.gz"
BC_LONGLIST_MULTIOME = BC_LONGLIST_DIR / "737K-arc-v1.txt.gz"
##### Set output location #####
OUTPUT_BASE = pathlib.Path(config.get("OUTPUT_BASE", "./output"))
###################
# READ ADAPTER SCAN FILES
###################
INGEST_DIR = OUTPUT_BASE / "{run_id}" / "ingest"
FOFN = str(INGEST_DIR / "fofn.txt")
CHUNKED_FASTQ_DIR = INGEST_DIR / "chunked_fastqs"
CHUNKED_FASTQS = str(CHUNKED_FASTQ_DIR / "{batch_id}.fastq")
ADAPTERS_DIR = OUTPUT_BASE / "{run_id}" / "adapters"
READ_CONFIG_CHUNKED = str(ADAPTERS_DIR / "{batch_id}.configs.tsv")
STRANDED_FQ_CHUNKED = str(ADAPTERS_DIR / "{batch_id}.stranded.fastq")
READ_CONFIG = str(ADAPTERS_DIR / "configs.tsv")
STRANDED_FQ = str(ADAPTERS_DIR / "reads.stranded.fastq.gz")
CONFIG_STATS = str(ADAPTERS_DIR / "configs.stats.json")
###################
# REF ALIGN FILES
###################
REF_GENES_DIR = REF_GENES_GTF.parents[0]
REF_GENES_BED = str(REF_GENES_DIR / f'{REF_GENES_GTF.name.replace(".gtf", ".bed")}')
ALIGN_DIR = OUTPUT_BASE / "{run_id}" / "align"
REF_CHROM_SIZES = str(ALIGN_DIR / "chrom_sizes.tsv")
SAM_TMP = str(ALIGN_DIR / "tmp.sam")
BAM_UNSORT_TMP = str(ALIGN_DIR / "tmp.unsort.bam")
BAM_SORT = str(ALIGN_DIR / "sorted.bam")
BAM_SORT_BAI = str(ALIGN_DIR / "sorted.bam.bai")
###################
# BARCODE + UMI DEMUX FILES
###################
DEMUX_DIR = OUTPUT_BASE / "{run_id}" / "demux"
# All chrom files
BAM_BC_UNCORR_TMP = str(DEMUX_DIR / "bc_extract.tmp.sorted.bam")
BAM_BC_UNCORR = str(DEMUX_DIR / "bc_extract.sorted.bam")
BAM_BC_UNCORR_BAI = str(DEMUX_DIR / "bc_extract.sorted.bam.bai")
BARCODE_COUNTS = str(DEMUX_DIR / "uncorrected_bc_counts.tsv")
BARCODE_WHITELIST = str(DEMUX_DIR / "whitelist.tsv")
BARCODE_KNEEPLOT = str(DEMUX_DIR / "kneeplot.png")
# Chrom-specific files
SPLIT_DIR = DEMUX_DIR / "chroms"
CHROM_BAM_BC_UNCORR = str(SPLIT_DIR / "{chrom}.sorted.bam")
CHROM_BAM_BC_UNCORR_BAI = str(SPLIT_DIR / "{chrom}.sorted.bam.bai")
CHROM_BAM_BC_TMP = str(SPLIT_DIR / "{chrom}.bc_assign.tmp.bam")
CHROM_BAM_BC = str(SPLIT_DIR / "{chrom}.bc_assign.bam")
CHROM_BAM_BC_BAI = str(SPLIT_DIR / "{chrom}.bc_assign.bam.bai")
CHROM_ASSIGNED_BARCODE_COUNTS = str(SPLIT_DIR / "{chrom}.bc_assign_counts.tsv")
CHROM_BED_BC = str(SPLIT_DIR / "{chrom}.bc_assign.bed")
SPLIT_ANNOT_DIR = DEMUX_DIR / "refs"
CHROM_GTF = str(SPLIT_ANNOT_DIR / "{chrom}.genes.gtf")
CHROM_TSV_GENE_ASSIGNS = str(SPLIT_DIR / "{chrom}.read.gene_assigns.tsv")
CHROM_BAM_BC_GENE_TMP = str(SPLIT_DIR / "{chrom}.bc_assign.gene.tmp.bam")
CHROM_BAM_BC_GENE = str(SPLIT_DIR / "{chrom}.bc_assign.gene.bam")
CHROM_BAM_BC_GENE_BAI = str(SPLIT_DIR / "{chrom}.bc_assign.gene.bam.bai")
CHROM_BAM_FULLY_TAGGED_TMP = str(SPLIT_DIR / "{chrom}.tagged.tmp.bam")
CHROM_BAM_FULLY_TAGGED = str(SPLIT_DIR / "{chrom}.tagged.bam")
CHROM_BAM_FULLY_TAGGED_BAI = str(SPLIT_DIR / "{chrom}.tagged.bam.bai")
# Merged files
BAM_FULLY_TAGGED = str(DEMUX_DIR / "tagged.sorted.bam")
BAM_FULLY_TAGGED_BAI = str(DEMUX_DIR / "tagged.sorted.bam.bai")
ASSIGNED_BARCODE_COUNTS = str(DEMUX_DIR / "assigned_bc_counts.tsv")
CELL_UMI_GENE_TSV = str(DEMUX_DIR / "cell_umi_gene.tsv")
###################
# GENE EXPRESSION FILES
###################
MATRIX_DIR = OUTPUT_BASE / "{run_id}" / "matrix"
MATRIX_COUNTS_TSV = str(MATRIX_DIR / "gene_expression.counts.tsv")
MATRIX_MITO_TSV = str(MATRIX_DIR / "gene_expression.mito.tsv")
MATRIX_PROCESSED_TSV = str(MATRIX_DIR / "gene_expression.processed.tsv")
MATRIX_UMAP_TSV = str(MATRIX_DIR / "gene_expression.umap.tsv")
MATRIX_UMAP_PLOT_GENE = str(MATRIX_DIR / "umap.gene.{plot_gene}.png")
MATRIX_UMAP_PLOT_MITO = str(MATRIX_DIR / "umap.mitochondrial.png")
MATRIX_UMAP_PLOT_TOTAL = str(MATRIX_DIR / "umap.total.png")
###################
# LIBRARY SATURATION FILES
###################
SAT_DIR = OUTPUT_BASE / "{run_id}" / "saturation"
SAT_PLOT = str(SAT_DIR / "saturation_curves.png")
##### include rules #####
include: "rules/stranding.smk"
include: "rules/align.smk"
include: "rules/process_bams.smk"
wildcard_constraints:
chrom=r"\w+(\.\d+)?",
##### target rules #####
rule all:
input:
expand(CONFIG_STATS, run_id=RUN_IDS),
expand(MATRIX_UMAP_TSV, run_id=RUN_IDS),
expand(SAT_PLOT, run_id=RUN_IDS),
expand(MATRIX_UMAP_PLOT_TOTAL, run_id=RUN_IDS),
expand(MATRIX_UMAP_PLOT_MITO, run_id=RUN_IDS),
expand(MATRIX_UMAP_PLOT_GENE, run_id=RUN_IDS, plot_gene=PLOT_GENES),