diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/Snakefile b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/Snakefile new file mode 100644 index 0000000..6b5cd5a --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/Snakefile @@ -0,0 +1,350 @@ +# input your common thread(s) here + +# ALL: one rule to rule them all.. +# Important!: to run this entire make of snake, the input to 'all' should be equivalent to the **final** filename at the end of the pipe +# Note: if you want to run a partial pipeline you can add an input for 'all' here that is equivalent to the output of a specific chunk (e.g. mapping) +rule all: + # remember - 4 spaces via python syntax! + input: + # -remember - 8 spaces via pyton syntax! DO NOT LEAVE EMPTY LINES FOR INPUT OR OUTPUT + # RAW QC - check for upload succes via md5checksum + "rawQC", + # trim 'adapters only' - currently by (i) adapter fasta and (ii) by quality score + "fastp_just_adapters", + # trim 'clean' - including additional complexity calls to omit poly-A tails (TagSeq) + "fastp_clean_n_pristine", + # map clean reads to the current GenBank genome + "hisat2_bamified", + # stringtie2 + "stringtie2_done", + # prep.py to output gene abundnace matrix + "complete" +# Initiate symbolic dirs to raw data - run raw QC - output multiQC report +rule rawQC: + #input: + # insert here + output: + # insert here + "rawQC" + #"md5checksum_complete" + shell: + """ + echo 'start' $(date) + + # RAW QC + + # load modules + module load bio/fastp/0.23.2 + module load bio/fastqc/0.11.9 + + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/raw # make directory for trimmed fastq files and multiqc report + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/ + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/rawqc + + # call dirs + BASEDIR=~/Airradians_F2Juveniles_TagSeq + RAWSEQ=~/../../../share/nefsc/mcfarland_sequecenes/TagSeq_F2Juvenile_scallops_RTLGurr7206 + DATDIR=~/Airradians_F2Juveniles_TagSeq/raw + PYTHONENV=~/python_venv/bin + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/rawqc + + # nav to dir for symbolic links + cd $DATDIR + + # symbolically link clean reads to fastp_multiQC dir + ln -s $RAWSEQ/*.fastq.gz ./ # call backward from the directory to the share folder, input symbolic links to 'raw' folder as $DATDIR + + # make an array of sequences to trim + array=($(ls $DATDIR/*.fastq.gz)) # call the folder will all symbolically linked .fastq.gz files (without the SA* folder included) + + # fastqc loop of raw reads - output fastqc files to raw folder + for i in ${{array[@]}}; do + fastqc ${{i}} --outdir $OUTDIR; + done + + echo "QC of raw reads complete." $(date) + + # activate python + source $PYTHONENV/activate # from the current directory, activates the bin of installed python packages, including multiqc + + # mutliqc compiling all contents within the output folder + multiqc $OUTDIR -o $OUTDIR #Compile MultiQC report from FastQC files - output .html in current directory ( multiQC html report) + + # exit python virtual envrionment + #deactivate + + # out file + touch $BASEDIR/rawQC + + echo 'end' $(date) + """ +# trim just adapters + phred 30 - output mutliqc report +rule trim_adapters: + input: + "rawQC" + output: + "fastp_just_adapters" + shell: + """ + echo 'start' $(date) + + # Trim using fastp (adapters + phred only) + + # load modules needed + module load bio/fastp/0.23.2 + module load bio/fastqc/0.11.9 + + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/trim + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/trim/adapters_only + + # call dirs + BASEDIR=~/Airradians_F2Juveniles_TagSeq + DATDIR=~/Airradians_F2Juveniles_TagSeq/raw + PYTHONENV=~/python_venv/bin + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/trim/adapters_only + + # nav to data dir + cd $DATDIR + + # Make an array of sequences to trim + array=($(ls *.fastq.gz)) # call symbolically linked .fastq files + + # fastp loop; trim the Read 1 TruSeq adapter sequence; trim poly x default 10 (to trim polyA) + for i in ${{array[@]}}; do + fastp --in1 ${{i}} --out1 $OUTDIR/adapters.${{i}} --adapter_fasta $BASEDIR/adapters.fasta -q 30 # trim phred score + fastqc $OUTDIR/adapters.${{i}} --outdir $OUTDIR # calls the files output by fastp in previous line and output into the same folder + done; + + echo "Read trimming of adapters complete." $(date) + + # activate python + #source $PYTHONENV/activate # from the current directory, activates the bin of installed python packages, including multic + + # navigate to all the outdir files + cd $OUTDIR + + # mutliqc compiling all contents within the output folder + multiqc $OUTDIR -o $OUTDIR #Compile MultiQC report from FastQC files - output .html in current directory ( multiQC html report) + + # exit python virtual envrionment + #deactivate + + echo "Clean MultiQC report generated." $(date) + + # out file + touch $BASEDIR/fastp_just_adapters + + echo 'end' $(date) + """ +# trim just adapters + phred 30 - output mutliqc report +rule trim_clean: + input: + "fastp_just_adapters" + output: + "fastp_clean_n_pristine" + shell: + """ + echo 'start' $(date) + + # Trim using fastp (adapters + phred + poly-A tail) + + # load modules needed + module load bio/fastp/0.23.2 + module load bio/fastqc/0.11.9 + + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/trim + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/trim/clean + + # call dirs + BASEDIR=~/Airradians_F2Juveniles_TagSeq + DATDIR=~/Airradians_F2Juveniles_TagSeq/raw + PYTHONENV=~/python_venv/bin + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/trim/clean + + # nav to data dir + cd $DATDIR + + # Make an array of sequences to trim + array=($(ls *.fastq.gz)) # call the folder will all symbolically linked .fastq.gz files + + # fastp loop; trim the Read 1 TruSeq adapter sequence; trim poly x default 10 (to trim polyA) + for i in ${{array[@]}}; do + fastp --in1 ${{i}} --out1 $OUTDIR/clean.${{i}} --adapter_fasta $BASEDIR/adapters.fasta --trim_poly_x 6 -q 30 -y -Y 50 # trim poly to remove poly A tail, -q to trim phred score, -y -Y 50 for seq complexity (default 30) + fastqc $OUTDIR/clean.${{i}} --outdir $OUTDIR # calls the files output by fastp in previous line and output into the same folder + done + + echo "Read trimming of adapters complete." $(date) + + # activate python + source $PYTHONENV/activate # from the current directory, activates the bin of installed python packages, including multiqc + + # navigate to all the outdir files + cd $OUTDIR + + # autliqc compiling all contents within the output folder + multiqc $OUTDIR -o $OUTDIR #Compile MultiQC report from FastQC files - output .html in current directory ( multiQC html report) + + # exit python virtual envrionment + #deactivate + + echo "Clean MultiQC report generated." $(date) + + # out file + touch $BASEDIR/fastp_clean_n_prisitine + + echo 'end' $(date) + """ +# map clean reads from 'trim_clean' to the current reference genome using hisat2 +rule map: + input: + "fastp_clean_n_pristine" + output: + "hisat2_bamified" + shell: + """ + echo 'start' $(date) + + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/hisat2/ + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map + + # call dirs + BASEDIR=~/Airradians_F2Juveniles_TagSeq + REFDIR=~/refs # the reference folder + PYTHONENV=~/python_venv/bin # python enrnvrionment + DATDIR=~/Airradians_F2Juveniles_TagSeq/output/trim/clean # directory of trimmed and filtered fastq.gz files + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map + + # load modules, requires hisat2 and samtools + module load bio/hisat2/2.2.1 + module load bio/samtools/1.11 + + # symbolically link clean reads to hisat2 dir + ln -s $DATDIR/*.fastq.gz $OUTDIR/ # call the .fastq.gz output from fastp trim - make symb link to output/hisat2 + + #echo "Symbolic directories successfully linked" + + # activate python for hisat2-build + source $PYTHONENV/activate # activate python virtual envriomment to call python and run hisat2-build + + #echo "Python virtual env activated" + + # index the reference genome for Panopea generosa output index to working directory + #hisat2-build -f $REFDIR/Argopecten_irradians_irradians_genome.fasta $OUTDIR/Airradians_ref # old reference genome + #hisat2-build -f $REFDIR/GCF_041381155.1_Ai_NY_cds_from_genomic.fna $OUTDIR/Airradians_ref + hisat2-build -f $REFDIR/GCF_041381155.1_Ai_NY_genomic.fna $OUTDIR/Airradians_ref + + #echo "Referece genome indexed. Starting alingment" $(date) + + # exit python virtual envrionment + #deactivate + + # nav to out dir + cd $OUTDIR + + # This script exports alignments as bam files + # sorts the bam file because Stringtie takes a sorted file for input (--dta) + # removes the sam file because it is no longer needed + array=($(ls ./clean*.fastq.gz)) # call the symbolically linked sequences - make an array to align + for i in ${{array[@]}}; do + hisat2 -p 8 --dta -x $OUTDIR/Airradians_ref -U ${{i}} -S ${{i}}.sam + samtools sort -@ 8 -o ${{i}}.bam ${{i}}.sam + echo "${{i}} bam-ified!" + rm ${{i}}.sam + done + + # out file + touch $BASEDIR/hisat2_bamified + + echo 'end' $(date) + """ +rule stringtie2: + input: + "hisat2_bamified" + output: + "stringtie2_done" + shell: + """ + echo 'start' $(date) + + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/stringtie2 + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/stringtie2/Airradians + + # call dirs + BASEDIR=~/Airradians_F2Juveniles_TagSeq # base directory + REFDIR=~/refs + DATDIR=~/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map # directory of mapped bam files from hisat2 + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/stringtie2/ + + # load modules, requires stringtie2 + module load bio/stringtie/2.2.0 + + array=($(ls ${{DATDIR}}/*.bam)) #Make an array of sequences to assemble + + # awk example: file name clean.C9-larva-22_S77.bam - use awk to get sample name w/o .bam using . as delimiter + # run strnigtie on the array and output to the stringtie2 directory + for i in ${{array[@]}}; do #Running with the -e option to compare output to exclude novel genes. Also output a file with the gene abundances + sample_name=`echo ${{i}}| awk -F [..] '{{print $2}}'` + #stringtie -p 8 -e -B -G $REFDIR/Argopecten_irradians_irradians.gff -A $OUTDIR/${{sample_name}}.gene_abund.tab -o $OUTDIR/${{sample_name}}.gtf ${{i}} # old reference + stringtie -p 8 -e -B -G $REFDIR/GCF_041381155.1_Ai_NY_genomic.gff -A $OUTDIR/${{sample_name}}.gene_abund.tab -o $OUTDIR/${{sample_name}}.gtf ${{i}} + done + + #out file + touch $BASEDIR/stringtie2_done + + echo "StringTie assembly COMPLETE, starting assembly analysis" $(date) + """ +rule abundance_matrix: + input: + "stringtie2_done" + output: + "complete" + shell: + """ + # make dirs + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/stringtie2/Airradians/merged + mkdir -p ~/Airradians_F2Juveniles_TagSeq/output/count_matrix + + # load packages + module load bio/stringtie/2.2.0 + module load bio/gffcompare/0.12.6 + + # call command names + BASEDIR=~/Airradians_F2Juveniles_TagSeq # base directory + REFDIR=~/refs # direcrory with ref genome data + DATDIR=~/Airradians_F2Juveniles_TagSeq/output/stringtie2/ # outdir of the gtf stringtie2 files in stringtie2.sh + OUTDIR=~/Airradians_F2Juveniles_TagSeq/output/count_matrix # output of prepde.py + SCRIPTS=~/Airradians_F2Juveniles_TagSeq/scripts + + cd $DATDIR # to strngtie2 gtf outputs in previous chunk + + # gtf_list.txt and listGTF.txt + ls *.gtf > gtf_list.txt # list the .gtf files in output/stringtie2/Airradians + + # run stringtie merge + #stringtie --merge -p 8 $REFDIR/GCF_041381155.1_Ai_NY_genomic.gff -o Airradians_merged.gtf gtf_list.txt # merge GTFs in the $DATDIR directory + #echo "Stringtie merge complete" $(date) + + # run gff compaure to report accuracy of alignment to reference + #gffcompare -r $REFDIR/Argopecten_irradians_irradians.gff -G -o $DATDIR/merged $DATDIR/Airradians_merged.gtf #Compute the accuracy and pre$ + #echo "GFFcompare complete, Starting gene count matrix assembly..." $(date) + + + # list gtf files to call in preppy + for filename in ${{DATDIR}}/*.gtf; # call the merged files output in stringtie above + do echo $(basename -- $filename) $filename; + done > $DATDIR/listGTF.txt # full root to each .gtf file in output/stringtie2 - call in prepDE.py as -i + + # prepDE.py to assemble count matrix for R + # python is an alias for python_venv/bin/python (requires virtual envrionment installation on sedna) + python2 $SCRIPTS/prepDE.py3 -g $OUTDIR/Airradians_gene_count_matrix.csv -t $OUTDIR/Airradians_transcript_count_matrix.csv -i $DATDIR/listGTF.txt #Compile the gene count matrix + echo "Gene count matrix compiled." $(date) + + # out file + touch $BASEDIR/complete + """ diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/abundance_matrix/Airradians_gene_count_matrix.csv b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/abundance_matrix/Airradians_gene_count_matrix.csv new file mode 100644 index 0000000..e569828 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/abundance_matrix/Airradians_gene_count_matrix.csv @@ -0,0 +1,26367 @@ +gene_id,7206-B11-HiSeqTranscript_S4_R1_001.gtf,7206-B11-HiSeqTranscript_S4_R2_001.gtf,7206-B12-HiSeqTranscript_S5_R1_001.gtf,7206-B12-HiSeqTranscript_S5_R2_001.gtf,7206-C1-HiSeqTranscript_S1_R1_001.gtf,7206-C1-HiSeqTranscript_S1_R2_001.gtf,7206-C11-HiSeqTranscript_S7_R1_001.gtf,7206-C11-HiSeqTranscript_S7_R2_001.gtf,7206-C12-HiSeqTranscript_S4_R1_001.gtf,7206-C12-HiSeqTranscript_S4_R2_001.gtf,7206-C2-HiSeqTranscript_S5_R1_001.gtf,7206-C2-HiSeqTranscript_S5_R2_001.gtf,7206-C3-HiSeqTranscript_S5_R1_001.gtf,7206-C3-HiSeqTranscript_S5_R2_001.gtf,7206-C4-HiSeqTranscript_S8_R1_001.gtf,7206-C4-HiSeqTranscript_S8_R2_001.gtf,7206-C5-HiSeqTranscript_S9_R1_001.gtf,7206-C5-HiSeqTranscript_S9_R2_001.gtf,7206-C6-HiSeqTranscript_S4_R1_001.gtf,7206-C6-HiSeqTranscript_S4_R2_001.gtf,7206-C7-HiSeqTranscript_S3_R1_001.gtf,7206-C7-HiSeqTranscript_S3_R2_001.gtf,7206-C8-HiSeqTranscript_S4_R1_001.gtf,7206-C8-HiSeqTranscript_S4_R2_001.gtf,7206-C9-HiSeqTranscript_S2_R1_001.gtf,7206-C9-HiSeqTranscript_S2_R2_001.gtf,7206-D1-HiSeqTranscript_S5_R1_001.gtf,7206-D1-HiSeqTranscript_S5_R2_001.gtf,7206-D2-HiSeqTranscript_S5_R1_001.gtf,7206-D2-HiSeqTranscript_S5_R2_001.gtf,7206-D3-HiSeqTranscript_S3_R1_001.gtf,7206-D3-HiSeqTranscript_S3_R2_001.gtf,7206-D4-HiSeqTranscript_S38_R1_001.gtf,7206-D4-HiSeqTranscript_S38_R2_001.gtf,7206-D5-HiSeqTranscript_S37_R1_001.gtf,7206-D5-HiSeqTranscript_S37_R2_001.gtf,7206-D6-HiSeqTranscript_S1_R1_001.gtf,7206-D6-HiSeqTranscript_S1_R2_001.gtf,7206-D7-HiSeqTranscript_S2_R1_001.gtf,7206-D7-HiSeqTranscript_S2_R2_001.gtf,7206-D8-HiSeqTranscript_S10_R1_001.gtf,7206-D8-HiSeqTranscript_S10_R2_001.gtf,7206-D9-HiSeqTranscript_S3_R1_001.gtf,7206-D9-HiSeqTranscript_S3_R2_001.gtf 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+rna-XM_069255322.1,0,0,0,0,476,521,0,0,0,381,0,0,0,0,0,246,150,163,0,394,483,434,381,335,332,410,0,0,997,1828,708,366,0,0,288,373,1104,895,687,659,464,0,595,691 +rna-XR_011209964.1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0 +gene-LOC138306547,,0,0,0,41,46,50,50,0,0,0,0,55,59,0,0,0,0,60,67,0,0,0,0,41,65,0,0,129,125,59,49,0,0,0,0,68,92,0,0,0,0,56,49 +gene-LOC138306546,,,,,,,,,26,22,0,0,16,23,15,16,0,0,0,0,0,0,0,0,0,0,0,0,60,59,11,9,0,0,17,19,17,18,0,10,0,0,0,10 +rna-XM_069245204.1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0 diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/config.yml b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/config.yml new file mode 100644 index 0000000..39ba1b6 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/config.yml @@ -0,0 +1,16 @@ +# cluster_config.yml - cluster configuration +__default__: + time: 10:00:00 + nodes: 1 + ntasks-per-node: 1 + mem: 80GB +# can modify here for customized err and out +trim_adapters: + ntasks-per-node: 9 + mem: 80GB +trim_clean: + ntasks-per-node: 9 + mem: 80GB +map: + ntasks-per-node: 12 + mem: 80GB diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/percent_mapped_F2 b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/percent_mapped_F2 new file mode 100644 index 0000000..4c3f032 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/percent_mapped_F2 @@ -0,0 +1,44 @@ +273:23.67% overall alignment rate +281:23.53% overall alignment rate +289:69.12% overall alignment rate +297:67.29% overall alignment rate +305:68.38% overall alignment rate +313:67.32% overall alignment rate +321:66.54% overall alignment rate +329:66.06% overall alignment rate +337:67.69% overall alignment rate +345:69.08% overall alignment rate +353:44.42% overall alignment rate +361:44.25% overall alignment rate +369:65.90% overall alignment rate +377:67.24% overall alignment rate +385:68.50% overall alignment rate +393:68.22% overall alignment rate +401:67.27% overall alignment rate +409:66.87% overall alignment rate +417:67.03% overall alignment rate +425:68.61% overall alignment rate +433:68.02% overall alignment rate +441:67.04% overall alignment rate +449:66.72% overall alignment rate +457:65.68% overall alignment rate +465:67.63% overall alignment rate +473:69.37% overall alignment rate +481:66.07% overall alignment rate +489:65.59% overall alignment rate +497:69.60% overall alignment rate +505:69.18% overall alignment rate +513:69.70% overall alignment rate +521:69.00% overall alignment rate +529:60.31% overall alignment rate +537:60.06% overall alignment rate +545:72.69% overall alignment rate +553:72.03% overall alignment rate +561:72.07% overall alignment rate +569:71.64% overall alignment rate +577:57.97% overall alignment rate +585:57.67% overall alignment rate +593:64.83% overall alignment rate +601:64.33% overall alignment rate +609:69.97% overall alignment rate +617:71.81% overall alignment rate diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/slurm_hisatmapping_outfile b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/slurm_hisatmapping_outfile new file mode 100644 index 0000000..2a23151 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/mapping_summary/slurm_hisatmapping_outfile @@ -0,0 +1,623 @@ +Building DAG of jobs... +Using shell: /usr/bin/bash +Provided cores: 12 +Rules claiming more threads will be scaled down. +Provided resources: mem_mb=1000, mem_mib=954, disk_mb=1000, disk_mib=954 +Select jobs to execute... + +[Thu Nov 14 19:17:23 2024] +rule map: + input: fastp_clean_n_pristine + output: hisat2_bamified + jobid: 0 + reason: Forced execution + resources: mem_mb=1000, mem_mib=954, disk_mb=1000, disk_mib=954, tmpdir=/tmp + +start Thu Nov 14 19:17:23 PST 2024 +Settings: + Output files: "/home/sgurr/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map/Airradians_ref.*.ht2" + Line rate: 6 (line is 64 bytes) + Lines per side: 1 (side is 64 bytes) + Offset rate: 4 (one in 16) + FTable chars: 10 + Strings: unpacked + Local offset rate: 3 (one in 8) + Local fTable chars: 6 + Local sequence length: 57344 + Local sequence overlap between two consecutive indexes: 1024 + Endianness: little + Actual local endianness: little + Sanity checking: disabled + Assertions: disabled + Random seed: 0 + Sizeofs: void*:8, int:4, long:8, size_t:8 +Input files DNA, FASTA: + /home/sgurr/refs/GCF_041381155.1_Ai_NY_genomic.fna +Reading reference sizes + Time reading reference sizes: 00:00:07 +Calculating joined length +Writing header +Reserving space for joined string +Joining reference sequences + Time to join reference sequences: 00:00:04 + Time to read SNPs and splice sites: 00:00:00 +Using parameters --bmax 158603202 --dcv 1024 + Doing ahead-of-time memory usage test + Passed! Constructing with these parameters: --bmax 158603202 --dcv 1024 +Constructing suffix-array element generator +Building DifferenceCoverSample + Building sPrime + Building sPrimeOrder + V-Sorting samples + V-Sorting samples time: 00:00:17 + Allocating rank array + Ranking v-sort output + Ranking v-sort output time: 00:00:04 + Invoking Larsson-Sadakane on ranks + Invoking Larsson-Sadakane on ranks time: 00:00:08 + Sanity-checking and returning +Building samples +Reserving space for 12 sample suffixes +Generating random suffixes +QSorting 12 sample offsets, eliminating duplicates +QSorting sample offsets, eliminating duplicates time: 00:00:00 +Multikey QSorting 12 samples + (Using difference cover) + Multikey QSorting samples time: 00:00:00 +Calculating bucket sizes +Splitting and merging + Splitting and merging time: 00:00:00 +Split 2, merged 8; iterating... +Splitting and merging + Splitting and merging time: 00:00:00 +Split 1, merged 0; iterating... +Splitting and merging + Splitting and merging time: 00:00:00 +Avg bucket size: 1.05735e+08 (target: 158603201) +Converting suffix-array elements to index image +Allocating ftab, absorbFtab +Entering GFM loop +Getting block 1 of 8 + Reserving size (158603202) for bucket 1 + Calculating Z arrays for bucket 1 + Entering block accumulator loop for bucket 1: + bucket 1: 10% + bucket 1: 20% + bucket 1: 30% + bucket 1: 40% + bucket 1: 50% + bucket 1: 60% + bucket 1: 70% + bucket 1: 80% + bucket 1: 90% + bucket 1: 100% + Sorting block of length 136683489 for bucket 1 + (Using difference cover) + Sorting block time: 00:00:48 +Returning block of 136683490 for bucket 1 +Getting block 2 of 8 + Reserving size (158603202) for bucket 2 + Calculating Z arrays for bucket 2 + Entering block accumulator loop for bucket 2: + bucket 2: 10% + bucket 2: 20% + bucket 2: 30% + bucket 2: 40% + bucket 2: 50% + bucket 2: 60% + bucket 2: 70% + bucket 2: 80% + bucket 2: 90% + bucket 2: 100% + Sorting block of length 62908349 for bucket 2 + (Using difference cover) + Sorting block time: 00:00:21 +Returning block of 62908350 for bucket 2 +Getting block 3 of 8 + Reserving size (158603202) for bucket 3 + Calculating Z arrays for bucket 3 + Entering block accumulator loop for bucket 3: + bucket 3: 10% + bucket 3: 20% + bucket 3: 30% + bucket 3: 40% + bucket 3: 50% + bucket 3: 60% + bucket 3: 70% + bucket 3: 80% + bucket 3: 90% + bucket 3: 100% + Sorting block of length 147167544 for bucket 3 + (Using difference cover) + Sorting block time: 00:00:51 +Returning block of 147167545 for bucket 3 +Getting block 4 of 8 + Reserving size (158603202) for bucket 4 + Calculating Z arrays for bucket 4 + Entering block accumulator loop for bucket 4: + bucket 4: 10% + bucket 4: 20% + bucket 4: 30% + bucket 4: 40% + bucket 4: 50% + bucket 4: 60% + bucket 4: 70% + bucket 4: 80% + bucket 4: 90% + bucket 4: 100% + Sorting block of length 115787003 for bucket 4 + (Using difference cover) + Sorting block time: 00:00:41 +Returning block of 115787004 for bucket 4 +Getting block 5 of 8 + Reserving size (158603202) for bucket 5 + Calculating Z arrays for bucket 5 + Entering block accumulator loop for bucket 5: + bucket 5: 10% + bucket 5: 20% + bucket 5: 30% + bucket 5: 40% + bucket 5: 50% + bucket 5: 60% + bucket 5: 70% + bucket 5: 80% + bucket 5: 90% + bucket 5: 100% + Sorting block of length 148784806 for bucket 5 + (Using difference cover) + Sorting block time: 00:00:52 +Returning block of 148784807 for bucket 5 +Getting block 6 of 8 + Reserving size (158603202) for bucket 6 + Calculating Z arrays for bucket 6 + Entering block accumulator loop for bucket 6: + bucket 6: 10% + bucket 6: 20% + bucket 6: 30% + bucket 6: 40% + bucket 6: 50% + bucket 6: 60% + bucket 6: 70% + bucket 6: 80% + bucket 6: 90% + bucket 6: 100% + Sorting block of length 66797307 for bucket 6 + (Using difference cover) + Sorting block time: 00:00:22 +Returning block of 66797308 for bucket 6 +Getting block 7 of 8 + Reserving size (158603202) for bucket 7 + Calculating Z arrays for bucket 7 + Entering block accumulator loop for bucket 7: + bucket 7: 10% + bucket 7: 20% + bucket 7: 30% + bucket 7: 40% + bucket 7: 50% + bucket 7: 60% + bucket 7: 70% + bucket 7: 80% + bucket 7: 90% + bucket 7: 100% + Sorting block of length 101077962 for bucket 7 + (Using difference cover) + Sorting block time: 00:00:34 +Returning block of 101077963 for bucket 7 +Getting block 8 of 8 + Reserving size (158603202) for bucket 8 + Calculating Z arrays for bucket 8 + Entering block accumulator loop for bucket 8: + bucket 8: 10% + bucket 8: 20% + bucket 8: 30% + bucket 8: 40% + bucket 8: 50% + bucket 8: 60% + bucket 8: 70% + bucket 8: 80% + bucket 8: 90% + bucket 8: 100% + Sorting block of length 66677280 for bucket 8 + (Using difference cover) + Sorting block time: 00:00:22 +Returning block of 66677281 for bucket 8 +Exited GFM loop +fchr[A]: 0 +fchr[C]: 272160501 +fchr[G]: 423006423 +fchr[T]: 573812258 +fchr[$]: 845883747 +Exiting GFM::buildToDisk() +Returning from initFromVector +Wrote 286372800 bytes to primary GFM file: /home/sgurr/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map/Airradians_ref.1.ht2 +Wrote 211470944 bytes to secondary GFM file: /home/sgurr/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map/Airradians_ref.2.ht2 +Re-opening _in1 and _in2 as input streams +Returning from GFM constructor +Returning from initFromVector +Wrote 378596871 bytes to primary GFM file: /home/sgurr/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map/Airradians_ref.5.ht2 +Wrote 215193418 bytes to secondary GFM file: /home/sgurr/Airradians_F2Juveniles_TagSeq/output/hisat2/Airradians_map/Airradians_ref.6.ht2 +Re-opening _in5 and _in5 as input streams +Returning from HGFM constructor +Headers: + len: 845883747 + gbwtLen: 845883748 + nodes: 845883748 + sz: 211470937 + gbwtSz: 211470938 + lineRate: 6 + offRate: 4 + offMask: 0xfffffff0 + ftabChars: 10 + eftabLen: 0 + eftabSz: 0 + ftabLen: 1048577 + ftabSz: 4194308 + offsLen: 52867735 + offsSz: 211470940 + lineSz: 64 + sideSz: 64 + sideGbwtSz: 48 + sideGbwtLen: 192 + numSides: 4405645 + numLines: 4405645 + gbwtTotLen: 281961280 + gbwtTotSz: 281961280 + reverse: 0 + linearFM: Yes +Total time for call to driver() for forward index: 00:11:59 +5773749 reads; of these: + 5773749 (100.00%) were unpaired; of these: + 4407242 (76.33%) aligned 0 times + 1243971 (21.55%) aligned exactly 1 time + 122536 (2.12%) aligned >1 times +23.67% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-B11-HiSeqTranscript_S4_R1_001.fastq.gz bam-ified! +5489480 reads; of these: + 5489480 (100.00%) were unpaired; of these: + 4197872 (76.47%) aligned 0 times + 1177101 (21.44%) aligned exactly 1 time + 114507 (2.09%) aligned >1 times +23.53% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-B11-HiSeqTranscript_S4_R2_001.fastq.gz bam-ified! +4827089 reads; of these: + 4827089 (100.00%) were unpaired; of these: + 1490746 (30.88%) aligned 0 times + 3163343 (65.53%) aligned exactly 1 time + 173000 (3.58%) aligned >1 times +69.12% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-B12-HiSeqTranscript_S5_R1_001.fastq.gz bam-ified! +4393127 reads; of these: + 4393127 (100.00%) were unpaired; of these: + 1436842 (32.71%) aligned 0 times + 2806255 (63.88%) aligned exactly 1 time + 150030 (3.42%) aligned >1 times +67.29% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-B12-HiSeqTranscript_S5_R2_001.fastq.gz bam-ified! +5926451 reads; of these: + 5926451 (100.00%) were unpaired; of these: + 1874214 (31.62%) aligned 0 times + 3804332 (64.19%) aligned exactly 1 time + 247905 (4.18%) aligned >1 times +68.38% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C11-HiSeqTranscript_S7_R1_001.fastq.gz bam-ified! +5561313 reads; of these: + 5561313 (100.00%) were unpaired; of these: + 1817647 (32.68%) aligned 0 times + 3519682 (63.29%) aligned exactly 1 time + 223984 (4.03%) aligned >1 times +67.32% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C11-HiSeqTranscript_S7_R2_001.fastq.gz bam-ified! +6017893 reads; of these: + 6017893 (100.00%) were unpaired; of these: + 2013300 (33.46%) aligned 0 times + 3778566 (62.79%) aligned exactly 1 time + 226027 (3.76%) aligned >1 times +66.54% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C12-HiSeqTranscript_S4_R1_001.fastq.gz bam-ified! +5812751 reads; of these: + 5812751 (100.00%) were unpaired; of these: + 1972948 (33.94%) aligned 0 times + 3624164 (62.35%) aligned exactly 1 time + 215639 (3.71%) aligned >1 times +66.06% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C12-HiSeqTranscript_S4_R2_001.fastq.gz bam-ified! +5276835 reads; of these: + 5276835 (100.00%) were unpaired; of these: + 1704915 (32.31%) aligned 0 times + 3371940 (63.90%) aligned exactly 1 time + 199980 (3.79%) aligned >1 times +67.69% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C1-HiSeqTranscript_S1_R1_001.fastq.gz bam-ified! +5461565 reads; of these: + 5461565 (100.00%) were unpaired; of these: + 1688752 (30.92%) aligned 0 times + 3557365 (65.13%) aligned exactly 1 time + 215448 (3.94%) aligned >1 times +69.08% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C1-HiSeqTranscript_S1_R2_001.fastq.gz bam-ified! +3493512 reads; of these: + 3493512 (100.00%) were unpaired; of these: + 1941814 (55.58%) aligned 0 times + 1474611 (42.21%) aligned exactly 1 time + 77087 (2.21%) aligned >1 times +44.42% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C2-HiSeqTranscript_S5_R1_001.fastq.gz bam-ified! +3387961 reads; of these: + 3387961 (100.00%) were unpaired; of these: + 1888697 (55.75%) aligned 0 times + 1425132 (42.06%) aligned exactly 1 time + 74132 (2.19%) aligned >1 times +44.25% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C2-HiSeqTranscript_S5_R2_001.fastq.gz bam-ified! +8118382 reads; of these: + 8118382 (100.00%) were unpaired; of these: + 2768757 (34.10%) aligned 0 times + 5081152 (62.59%) aligned exactly 1 time + 268473 (3.31%) aligned >1 times +65.90% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C3-HiSeqTranscript_S5_R1_001.fastq.gz bam-ified! +8649698 reads; of these: + 8649698 (100.00%) were unpaired; of these: + 2833955 (32.76%) aligned 0 times + 5518813 (63.80%) aligned exactly 1 time + 296930 (3.43%) aligned >1 times +67.24% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C3-HiSeqTranscript_S5_R2_001.fastq.gz bam-ified! +5623134 reads; of these: + 5623134 (100.00%) were unpaired; of these: + 1771351 (31.50%) aligned 0 times + 3658977 (65.07%) aligned exactly 1 time + 192806 (3.43%) aligned >1 times +68.50% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C4-HiSeqTranscript_S8_R1_001.fastq.gz bam-ified! +5464899 reads; of these: + 5464899 (100.00%) were unpaired; of these: + 1737004 (31.78%) aligned 0 times + 3541754 (64.81%) aligned exactly 1 time + 186141 (3.41%) aligned >1 times +68.22% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C4-HiSeqTranscript_S8_R2_001.fastq.gz bam-ified! +5333087 reads; of these: + 5333087 (100.00%) were unpaired; of these: + 1745394 (32.73%) aligned 0 times + 3380204 (63.38%) aligned exactly 1 time + 207489 (3.89%) aligned >1 times +67.27% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C5-HiSeqTranscript_S9_R1_001.fastq.gz bam-ified! +5128280 reads; of these: + 5128280 (100.00%) were unpaired; of these: + 1699158 (33.13%) aligned 0 times + 3232777 (63.04%) aligned exactly 1 time + 196345 (3.83%) aligned >1 times +66.87% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C5-HiSeqTranscript_S9_R2_001.fastq.gz bam-ified! +9703973 reads; of these: + 9703973 (100.00%) were unpaired; of these: + 3199315 (32.97%) aligned 0 times + 6113225 (63.00%) aligned exactly 1 time + 391433 (4.03%) aligned >1 times +67.03% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C6-HiSeqTranscript_S4_R1_001.fastq.gz bam-ified! +10238740 reads; of these: + 10238740 (100.00%) were unpaired; of these: + 3213838 (31.39%) aligned 0 times + 6593225 (64.39%) aligned exactly 1 time + 431677 (4.22%) aligned >1 times +68.61% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C6-HiSeqTranscript_S4_R2_001.fastq.gz bam-ified! +5177862 reads; of these: + 5177862 (100.00%) were unpaired; of these: + 1655690 (31.98%) aligned 0 times + 3336670 (64.44%) aligned exactly 1 time + 185502 (3.58%) aligned >1 times +68.02% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C7-HiSeqTranscript_S3_R1_001.fastq.gz bam-ified! +4841153 reads; of these: + 4841153 (100.00%) were unpaired; of these: + 1595651 (32.96%) aligned 0 times + 3079084 (63.60%) aligned exactly 1 time + 166418 (3.44%) aligned >1 times +67.04% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C7-HiSeqTranscript_S3_R2_001.fastq.gz bam-ified! +4986653 reads; of these: + 4986653 (100.00%) were unpaired; of these: + 1659322 (33.28%) aligned 0 times + 3131573 (62.80%) aligned exactly 1 time + 195758 (3.93%) aligned >1 times +66.72% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C8-HiSeqTranscript_S4_R1_001.fastq.gz bam-ified! +4676043 reads; of these: + 4676043 (100.00%) were unpaired; of these: + 1604795 (34.32%) aligned 0 times + 2893516 (61.88%) aligned exactly 1 time + 177732 (3.80%) aligned >1 times +65.68% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C8-HiSeqTranscript_S4_R2_001.fastq.gz bam-ified! +9704021 reads; of these: + 9704021 (100.00%) were unpaired; of these: + 3141380 (32.37%) aligned 0 times + 6118954 (63.06%) aligned exactly 1 time + 443687 (4.57%) aligned >1 times +67.63% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C9-HiSeqTranscript_S2_R1_001.fastq.gz bam-ified! +10271177 reads; of these: + 10271177 (100.00%) were unpaired; of these: + 3146084 (30.63%) aligned 0 times + 6627480 (64.53%) aligned exactly 1 time + 497613 (4.84%) aligned >1 times +69.37% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-C9-HiSeqTranscript_S2_R2_001.fastq.gz bam-ified! +5058403 reads; of these: + 5058403 (100.00%) were unpaired; of these: + 1716248 (33.93%) aligned 0 times + 3115064 (61.58%) aligned exactly 1 time + 227091 (4.49%) aligned >1 times +66.07% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D1-HiSeqTranscript_S5_R1_001.fastq.gz bam-ified! +4894848 reads; of these: + 4894848 (100.00%) were unpaired; of these: + 1684096 (34.41%) aligned 0 times + 2994535 (61.18%) aligned exactly 1 time + 216217 (4.42%) aligned >1 times +65.59% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D1-HiSeqTranscript_S5_R2_001.fastq.gz bam-ified! +23466080 reads; of these: + 23466080 (100.00%) were unpaired; of these: + 7132598 (30.40%) aligned 0 times + 15528576 (66.17%) aligned exactly 1 time + 804906 (3.43%) aligned >1 times +69.60% overall alignment rate +[bam_sort_core] merging from 8 files and 8 in-memory blocks... +./clean.7206-D2-HiSeqTranscript_S5_R1_001.fastq.gz bam-ified! +22780006 reads; of these: + 22780006 (100.00%) were unpaired; of these: + 7020508 (30.82%) aligned 0 times + 14992535 (65.81%) aligned exactly 1 time + 766963 (3.37%) aligned >1 times +69.18% overall alignment rate +[bam_sort_core] merging from 8 files and 8 in-memory blocks... +./clean.7206-D2-HiSeqTranscript_S5_R2_001.fastq.gz bam-ified! +7104808 reads; of these: + 7104808 (100.00%) were unpaired; of these: + 2152953 (30.30%) aligned 0 times + 4674327 (65.79%) aligned exactly 1 time + 277528 (3.91%) aligned >1 times +69.70% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D3-HiSeqTranscript_S3_R1_001.fastq.gz bam-ified! +6836683 reads; of these: + 6836683 (100.00%) were unpaired; of these: + 2119222 (31.00%) aligned 0 times + 4454874 (65.16%) aligned exactly 1 time + 262587 (3.84%) aligned >1 times +69.00% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D3-HiSeqTranscript_S3_R2_001.fastq.gz bam-ified! +5810066 reads; of these: + 5810066 (100.00%) were unpaired; of these: + 2306145 (39.69%) aligned 0 times + 3330860 (57.33%) aligned exactly 1 time + 173061 (2.98%) aligned >1 times +60.31% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D4-HiSeqTranscript_S38_R1_001.fastq.gz bam-ified! +5680439 reads; of these: + 5680439 (100.00%) were unpaired; of these: + 2268837 (39.94%) aligned 0 times + 3244841 (57.12%) aligned exactly 1 time + 166761 (2.94%) aligned >1 times +60.06% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D4-HiSeqTranscript_S38_R2_001.fastq.gz bam-ified! +5298558 reads; of these: + 5298558 (100.00%) were unpaired; of these: + 1446773 (27.31%) aligned 0 times + 3647176 (68.83%) aligned exactly 1 time + 204609 (3.86%) aligned >1 times +72.69% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D5-HiSeqTranscript_S37_R1_001.fastq.gz bam-ified! +5098612 reads; of these: + 5098612 (100.00%) were unpaired; of these: + 1426085 (27.97%) aligned 0 times + 3479352 (68.24%) aligned exactly 1 time + 193175 (3.79%) aligned >1 times +72.03% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D5-HiSeqTranscript_S37_R2_001.fastq.gz bam-ified! +9691535 reads; of these: + 9691535 (100.00%) were unpaired; of these: + 2707292 (27.93%) aligned 0 times + 6633249 (68.44%) aligned exactly 1 time + 350994 (3.62%) aligned >1 times +72.07% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D6-HiSeqTranscript_S1_R1_001.fastq.gz bam-ified! +9503457 reads; of these: + 9503457 (100.00%) were unpaired; of these: + 2695300 (28.36%) aligned 0 times + 6465845 (68.04%) aligned exactly 1 time + 342312 (3.60%) aligned >1 times +71.64% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D6-HiSeqTranscript_S1_R2_001.fastq.gz bam-ified! +9959648 reads; of these: + 9959648 (100.00%) were unpaired; of these: + 4186128 (42.03%) aligned 0 times + 5347259 (53.69%) aligned exactly 1 time + 426261 (4.28%) aligned >1 times +57.97% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D7-HiSeqTranscript_S2_R1_001.fastq.gz bam-ified! +9680506 reads; of these: + 9680506 (100.00%) were unpaired; of these: + 4097679 (42.33%) aligned 0 times + 5173999 (53.45%) aligned exactly 1 time + 408828 (4.22%) aligned >1 times +57.67% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D7-HiSeqTranscript_S2_R2_001.fastq.gz bam-ified! +4990238 reads; of these: + 4990238 (100.00%) were unpaired; of these: + 1755218 (35.17%) aligned 0 times + 3039221 (60.90%) aligned exactly 1 time + 195799 (3.92%) aligned >1 times +64.83% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D8-HiSeqTranscript_S10_R1_001.fastq.gz bam-ified! +4810885 reads; of these: + 4810885 (100.00%) were unpaired; of these: + 1715846 (35.67%) aligned 0 times + 2909031 (60.47%) aligned exactly 1 time + 186008 (3.87%) aligned >1 times +64.33% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D8-HiSeqTranscript_S10_R2_001.fastq.gz bam-ified! +5531707 reads; of these: + 5531707 (100.00%) were unpaired; of these: + 1661331 (30.03%) aligned 0 times + 3672229 (66.39%) aligned exactly 1 time + 198147 (3.58%) aligned >1 times +69.97% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D9-HiSeqTranscript_S3_R1_001.fastq.gz bam-ified! +5857923 reads; of these: + 5857923 (100.00%) were unpaired; of these: + 1651540 (28.19%) aligned 0 times + 3986347 (68.05%) aligned exactly 1 time + 220036 (3.76%) aligned >1 times +71.81% overall alignment rate +[bam_sort_core] merging from 0 files and 8 in-memory blocks... +./clean.7206-D9-HiSeqTranscript_S3_R2_001.fastq.gz bam-ified! +end Thu Nov 14 20:18:10 PST 2024 +[Thu Nov 14 20:18:10 2024] +Finished job 0. +1 of 1 steps (100%) done diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/prepDE.py3 b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/prepDE.py3 new file mode 100644 index 0000000..ab5ee66 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/prepDE.py3 @@ -0,0 +1,304 @@ +#!/usr/bin/env python3 +import re, csv, sys, os, glob, warnings, itertools +from math import ceil +from optparse import OptionParser +from operator import itemgetter +from collections import defaultdict + +parser=OptionParser(description='Generates two CSV files containing the count matrices for genes and transcripts, using the coverage values found in the output of `stringtie -e`') +parser.add_option('-i', '--input', '--in', default='.', help="a folder containing all sample sub-directories, or a text file with sample ID and path to its GTF file on each line [default: %default/]") +parser.add_option('-g', default='gene_count_matrix.csv', help="where to output the gene count matrix [default: %default") +parser.add_option('-t', default='transcript_count_matrix.csv', help="where to output the transcript count matrix [default: %default]") +parser.add_option('-l', '--length', default=75, type='int', help="the average read length [default: %default]") +parser.add_option('-p', '--pattern', default=".", help="a regular expression that selects the sample subdirectories") +parser.add_option('-c', '--cluster', action="store_true", help="whether to cluster genes that overlap with different gene IDs, ignoring ones with geneID pattern (see below)") +parser.add_option('-s', '--string', default="MSTRG", help="if a different prefix is used for geneIDs assigned by StringTie [default: %default]") +parser.add_option('-k', '--key', default="prepG", help="if clustering, what prefix to use for geneIDs assigned by this script [default: %default]") +parser.add_option('-v', action="store_true", help="enable verbose processing") + +parser.add_option('--legend', default="legend.csv", help="if clustering, where to output the legend file mapping transcripts to assigned geneIDs [default: %default]") +(opts, args)=parser.parse_args() + +samples = [] # List of tuples. If sample list, (first column, path). Else, (subdirectory name, path to gtf file in subdirectory) +if (os.path.isfile(opts.input)): + # gtfList = True + try: + fin = open(opts.input, 'r') + for line in fin: + if line[0] != '#': + lineLst = tuple(line.strip().split(None,2)) + if (len(lineLst) != 2): + print("Error: line should have a sample ID and a file path:\n%s" % (line.strip())) + exit(1) + if lineLst[0] in samples: + print("Error: non-unique sample ID (%s)" % (lineLst[0])) + exit(1) + if not os.path.isfile(lineLst[1]): + print("Error: GTF file not found (%s)" % (lineLst[1])) + exit(1) + samples.append(lineLst) + except IOError: + print("Error: List of .gtf files, %s, doesn't exist" % (opts.input)) + exit(1) +else: + # gtfList = False + ## Check that opts.input directory exists + if not os.path.isdir(opts.input): + parser.print_help() + print(" ") + print("Error: sub-directory '%s' not found!" % (opts.input)) + sys.exit(1) + ##### + ## Collect all samples file paths and if empty print help message and quit + ##### + samples = [] + for i in next(os.walk(opts.input))[1]: + if re.search(opts.pattern,i): + for f in glob.iglob(os.path.join(opts.input,i,"*.gtf")): + samples.append((i,f)) + +if len(samples) == 0: + parser.print_help() + print(" ") + print("Error: no GTF files found under base directory %s !" % (opts.input)) + sys.exit(1) + +RE_GENE_ID=re.compile('gene_id "([^"]+)"') +RE_GENE_NAME=re.compile('gene_name "([^"]+)"') +RE_TRANSCRIPT_ID=re.compile('transcript_id "([^"]+)"') +RE_COVERAGE=re.compile('cov "([\-\+\d\.]+)"') +RE_STRING=re.compile(re.escape(opts.string)) + +RE_GFILE=re.compile('\-G\s*(\S+)') #assume filepath without spaces.. + + +##### +## Sort the sample names by the sample ID +##### + +samples.sort() + +#if opts.v: +# print "Sample GTFs found:" +# for s in samples: +# print s[1] + +##### +## Checks whether a given row is a transcript +## other options: ex. exon, transcript, mRNA, 5'UTR +##### +def is_transcript(x): + return len(x)>2 and x[2]=="transcript" + +def getGeneID(s, ctg, tid): + r=RE_GENE_ID.search(s) + #if r: return r.group(1) + rn=RE_GENE_NAME.search(s) + #if rn: return ctg+'|'+rn.group(1) + if r: + if rn: + return r.group(1)+'|'+rn.group(1) + else: + return r.group(1) + return tid + +def getCov(s): + r=RE_COVERAGE.search(s) + if r: + v=float(r.group(1)) + if v<0.0: v=0.0 + return v + return 0.0 + +def is_overlap(x,y): #NEEDS TO BE INTS! + return x[0]<=y[1] and y[0]<=x[1] + + +def t_overlap(t1, t2): #from badGenes: chromosome, strand, cluster, start, end, (e1start, e1end)... + if t1[0] != t2[0] or t1[1] != t2[1] or t1[5]0: + clusters=[] #lists of lists (could be sets) or something of transcripts + badGenes.sort(key=itemgetter(3)) #sort by start coord...? + i=0 + while i1: + clusters.append([t[2] for t in temp_cluster]) + i+=1 + + print(len(clusters)) + + for c in clusters: + c.sort() + + clusters.sort(key=itemgetter(0)) + legend=[] + for u,c in enumerate(clusters): + my_ID=opts.key+str((u+1)) + legend.append(list(itertools.chain.from_iterable([[my_ID],c]))) #my_ID, clustered transcript IDs + for t in c: + geneIDs[t]=my_ID +## geneIDs[t]="|".join(c) #duct-tape transcript IDs together, disregarding ref_gene_names and things like that + + with open(opts.legend, 'w') as l_file: + my_writer=csv.writer(l_file) + my_writer.writerows(legend) + +geneDict=defaultdict(lambda: defaultdict(lambda: 0)) #key=gene/cluster, value=dictionary with key=sample, value=summed counts +t_dict=defaultdict(lambda: defaultdict(lambda: 0)) +guidesFile='' # file given with -G for the 1st sample +for q, s in enumerate(samples): + if opts.v: + print(">processing sample %s from file %s" % s) + lno=0 + try: + #with open(glob.iglob(os.path.join(opts.input,s,"*.gtf")).next()) as f: #grabs first .gtf file it finds inside the sample subdirectory +# if not gtfList: +# f = open(glob.iglob(os.path.join(opts.input,s[1],"*.gtf")).next()) +# else: + f = open(s[1]) + transcript_len=0 + + for l in f: + lno+=1 + if l.startswith('#'): + if lno==1: + ei=l.find('-e') + if ei<0: + print("Error: sample file %s was not generated with -e option!" % ( s[1] )) + sys.exit(1) + gf=RE_GFILE.search(l) + if gf: + gfile=gf.group(1) + if guidesFile: + if gfile != guidesFile: + print("Warning: sample file %s generated with a different -G file (%s) than the first sample (%s)" % ( s[1], gfile, guidesFile )) + else: + guidesFile=gfile + else: + print("Error: sample %s was not processed with -G option!" % ( s[1] )) + sys.exit(1) + continue + v=l.split('\t') + if v[2]=="transcript": + if transcript_len>0: +## transcriptList.append((g_id, t_id, int(ceil(coverage*transcript_len/read_len)))) + t_dict[t_id][s[0]] = int(ceil(coverage*transcript_len/read_len)) + t_id=RE_TRANSCRIPT_ID.search(v[len(v)-1]).group(1) + #g_id=RE_GENE_ID.search(v[len(v)-1]).group(1) + g_id=getGeneID(v[8], v[0], t_id) + #coverage=float(RE_COVERAGE.search(v[len(v)-1]).group(1)) + coverage=getCov(v[8]) + transcript_len=0 + if v[2]=="exon": + transcript_len+=int(v[4])-int(v[3])+1 #because end coordinates are inclusive in GTF + +## transcriptList.append((g_id, t_id, int(ceil(coverage*transcript_len/read_len)))) + t_dict[t_id][s[0]]=int(ceil(coverage*transcript_len/read_len)) + + except StopIteration: +# if not gtfList: +# warnings.warn("No GTF file found in " + os.path.join(opts.input,s[1])) +# else: + warnings.warn("No GTF file found in " + s[1]) + + +## transcriptList.sort(key=lambda bla: bla[1]) #gene_id + + for i,v in t_dict.items(): +## print i,v + try: + geneDict[geneIDs[i]][s[0]]+=v[s[0]] + except KeyError: + print("Error: could not locate transcript %s entry for sample %s" % ( i, s[0] )) + raise + +if opts.v: + print("..writing %s " % ( opts.t )) +with open(opts.t, 'w') as csvfile: + my_writer = csv.DictWriter(csvfile, fieldnames = ["transcript_id"] + [x for x,y in samples]) + my_writer.writerow(dict((fn,fn) for fn in my_writer.fieldnames)) + for i in t_dict: + t_dict[i]["transcript_id"] = i + my_writer.writerow(t_dict[i]) +if opts.v: + print("..writing %s " % ( opts.g )) +with open(opts.g, 'w') as csvfile: + my_writer = csv.DictWriter(csvfile, fieldnames = ["gene_id"] + [x for x,y in samples]) +## my_writer.writerow([""]+samples) +## my_writer.writerows(geneDict) + my_writer.writerow(dict((fn,fn) for fn in my_writer.fieldnames)) + for i in geneDict: + geneDict[i]["gene_id"] = i #add gene_id to row + my_writer.writerow(geneDict[i]) +if opts.v: + print("All done.") + diff --git a/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/snake_manager.sh b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/snake_manager.sh new file mode 100644 index 0000000..1cd2688 --- /dev/null +++ b/HPC_analysis/output/Transriptomics/TagSeq_F2_juveniles/snake_manager.sh @@ -0,0 +1,14 @@ +#!/bin/bash +#SBATCH -t 250:00:00 +#SBATCH --nodes=1 +#SBATCH --ntasks-per-node=12 +#SBATCH --cpus-per-task=1 +#SBATCH --export=NONE +#SBATCH --mail-user=samuel.gurr@noaa.gov +#SBATCH --output=./"%x_out.%j" +#SBATCH --error=./"%x_err.%j" + +source ~/.bashrc +mamba activate snakemake-7.32.4 +snakemake --unlock # in case the job was canceled prior, +snakemake --cluster "sbatch -t {cluster.time} -N {cluster.nodes} --ntasks-per-node {cluster.ntasks-per-node} --mem {cluster.mem}" --latency-wait 20 --cluster-config config.yml -j 10