def find_isoform(gff3, genome_bam, isoform_gff3, tss_tes_stat, genomefa,
transcript_fa, downsample_ratio, config_dict,
raw_splice_isoform):
# find isoform
print "#### Read genne annotations"
chr_to_gene, transcript_dict, gene_to_transcript, transcript_to_exon = parse_gff_tree(
gff3)
transcript_to_junctions = {
tr: blocks_to_junctions(transcript_to_exon[tr])
for tr in transcript_to_exon
}
remove_similar_tr(gene_to_transcript, transcript_to_exon)
gene_dict = get_gene_flat(gene_to_transcript, transcript_to_exon)
chr_to_blocks = get_gene_blocks(gene_dict, chr_to_gene, gene_to_transcript)
# finding isoforms are required
print "#### find isoforms"
group_bam2isoform(genome_bam,
isoform_gff3,
tss_tes_stat,
"",
chr_to_blocks,
gene_dict,
transcript_to_junctions,
transcript_dict,
genomefa,
config=config_dict["isoform_parameters"],
downsample_ratio=downsample_ratio,
raw_gff3=None)
#raw_gff3=raw_splice_isoform if config_dict["global_parameters"]["generate_raw_isoform"] else None)
# get fasta
#print "### generate transcript fasta file", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
chr_to_gene_i, transcript_dict_i, gene_to_transcript_i, transcript_to_exon_i = parse_gff_tree(
isoform_gff3)
ref_dict = {
"chr_to_gene": chr_to_gene,
"transcript_dict": transcript_dict,
"gene_to_transcript": gene_to_transcript,
"transcript_to_exon": transcript_to_exon
}
if not config_dict["realign_parameters"]["use_annotation"]:
ref_dict = None
get_transcript_seq(genomefa,
transcript_fa,
chr_to_gene_i,
transcript_dict_i,
gene_to_transcript_i,
transcript_to_exon_i,
ref_dict=ref_dict)
return {
"transcript_dict": transcript_dict,
"transcript_dict_i": transcript_dict_i
}
def sc_long_pipeline(args):
# parse configuration file
if os.path.isfile(args.config_file):
print("Use config file: {}".format(args.config_file))
config_dict = parse_json_config(args.config_file)
elif os.path.isfile(os.path.join(sys.path[0], args.config_file)):
print("Use config file: {}".format(os.path.join(sys.path[0], args.config_file)))
config_dict = parse_json_config(os.path.join(sys.path[0], args.config_file))
else:
print("Cannot find config file in current directory or script depository: {}".format(args.config_file))
exit()
print_config(config_dict)
# check if files exist
if args.downsample_ratio>1 or args.downsample_ratio<=0:
print("downsample_ratio shoulw between 0 and 1: {}".format(args.downsample_ratio))
exit()
if not (os.path.isfile(args.infq) and os.path.isfile(args.gff3) and os.path.isfile(args.genomefa)):
print("make sure all file exists:")
print(args.infq)
print(args.gff3)
print(args.genomefa)
exit()
if not os.path.exists(args.outdir):
os.makedirs(args.outdir)
print("output directory not exist, create one:")
print(args.outdir)
if args.inbam != "" and (not os.path.isfile(args.inbam)):
print("make sure input inbam file exists:")
print(args.inbam)
exit()
# output files:
isoform_gff3 = os.path.join(args.outdir, "isoform_annotated.gff3")
isoform_gff3_f = os.path.join(args.outdir, "isoform_annotated.filtered.gff3")
FSM_anno_out = os.path.join(args.outdir, "isoform_FSM_annotation.csv")
raw_splice_isoform = os.path.join(args.outdir, "splice_raw.gff3")
tss_tes_stat = os.path.join(args.outdir, "tss_tes.bedgraph")
transcript_fa = os.path.join(args.outdir, "transcript_assembly.fa")
transcript_fa_idx = os.path.join(args.outdir, "transcript_assembly.fa.fai")
tmp_bam = os.path.join(args.outdir, "tmp.align.bam")
tmp_bed = os.path.join(args.outdir, "tmp.splice_anno.bed12")
genome_bam = os.path.join(args.outdir, "align2genome.bam")
realign_bam = os.path.join(args.outdir, "realign2transcript.bam")
tr_cnt_csv = os.path.join(args.outdir, "transcript_count.csv.gz")
tr_badcov_cnt_csv = os.path.join(args.outdir, "transcript_count.bad_coverage.csv.gz")
print "Input parameters:"
print "\tgene annotation:", args.gff3
print "\tgenome fasta:", args.genomefa
if args.inbam != "":
print "\tinput bam:", args.inbam
genome_bam = args.inbam
else:
print "\tinput fastq:", args.infq
print "\toutput directory:", args.outdir
print "\tdirectory contains minimap2:", args.minimap2_dir
# align reads to genome
if args.inbam == "" and config_dict["pipeline_parameters"]["do_genome_alignment"]:
print "### align reads to genome using minimap2", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
if config_dict["alignment_parameters"]["use_junctions"]:
gff3_to_bed12(args.minimap2_dir, args.gff3, tmp_bed)
minimap2_align(args.minimap2_dir, args.genomefa, args.infq, tmp_bam, no_flank=config_dict["alignment_parameters"]["no_flank"], bed12_junc=tmp_bed if config_dict["alignment_parameters"]["use_junctions"] else None)
samtools_sort_index(tmp_bam, genome_bam)
os.remove(tmp_bam)
if config_dict["alignment_parameters"]["use_junctions"]:
os.remove(tmp_bed)
else:
print "### skip aligning reads to genome", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
# find isoform
print "### read gene annotation", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
chr_to_gene, transcript_dict, gene_to_transcript, transcript_to_exon = parse_gff_tree(args.gff3)
transcript_to_junctions = {tr: blocks_to_junctions(transcript_to_exon[tr]) for tr in transcript_to_exon}
remove_similar_tr(transcript_dict, gene_to_transcript, transcript_to_exon)
gene_dict = get_gene_flat(gene_to_transcript, transcript_to_exon)
chr_to_blocks = get_gene_blocks(gene_dict, chr_to_gene, gene_to_transcript)
if config_dict["pipeline_parameters"]["do_isoform_identification"]:
print "### find isoforms", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
group_bam2isoform(genome_bam, isoform_gff3, tss_tes_stat, "", chr_to_blocks, gene_dict, transcript_to_junctions, transcript_dict, args.genomefa,
config=config_dict["isoform_parameters"],
downsample_ratio=args.downsample_ratio,
raw_gff3=raw_splice_isoform if config_dict["global_parameters"]["generate_raw_isoform"] else None)
else:
print "### skip finding isoforms", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
# get fasta
#print "### generate transcript fasta file", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
chr_to_gene_i, transcript_dict_i, gene_to_transcript_i, transcript_to_exon_i = parse_gff_tree(isoform_gff3)
ref_dict = {"chr_to_gene":chr_to_gene, "transcript_dict":transcript_dict, "gene_to_transcript":gene_to_transcript, "transcript_to_exon":transcript_to_exon}
if not config_dict["realign_parameters"]["use_annotation"]:
ref_dict = None
get_transcript_seq(args.genomefa, transcript_fa, chr_to_gene_i, transcript_dict_i, gene_to_transcript_i, transcript_to_exon_i,ref_dict=ref_dict)
# realign to transcript using minimap2
if config_dict["pipeline_parameters"]["do_read_realignment"]:
print "### realign to transcript using minimap2", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
minimap2_tr_align(args.minimap2_dir, transcript_fa, args.infq, tmp_bam)
samtools_sort_index(tmp_bam, realign_bam)
os.remove(tmp_bam)
else:
print "### skip read realignment", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
# quantification
if config_dict["pipeline_parameters"]["do_transcript_quantification"]:
print "### generate transcript count matrix", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")
bc_tr_count_dict, bc_tr_badcov_count_dict, tr_kept = parse_realigned_bam(realign_bam, transcript_fa_idx, config_dict["isoform_parameters"]["Min_sup_cnt"], config_dict["transcript_counting"]["min_tr_coverage"], config_dict["transcript_counting"]["min_read_coverage"])
#realigned_bam_coverage(realign_bam, transcript_fa_idx, args.outdir)
tr_cnt = wrt_tr_to_csv(bc_tr_count_dict, transcript_dict_i, tr_cnt_csv, transcript_dict, config_dict["global_parameters"]["has_UMI"])
wrt_tr_to_csv(bc_tr_badcov_count_dict, transcript_dict_i, tr_badcov_cnt_csv, transcript_dict, config_dict["global_parameters"]["has_UMI"])
annotate_filter_gff(isoform_gff3,args.gff3,isoform_gff3_f,FSM_anno_out,tr_cnt,config_dict["isoform_parameters"]["Min_sup_cnt"])
else:
print "### skip transcript quantification", datetime.datetime.now().strftime("%Y-%m-%d %H:%M:%S")