GSE88722 Processing Pipeline

Publication

Methods (San Diego, Calif.) (2017) — PMID 28003131

Dataset

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

1. 1

   Takes output from raw files. Run to trim off both 5p™ and 3p™ adapters on both reads.

   cutadapt GitHub

   $ Bash example

   # Install cutadapt if not already installed
   # conda install -c bioconda cutadapt
   
   # Define adapter sequences (replace with actual sequences for your library prep)
   cutadapt quality-cutoff 6 -m 18 -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGT AGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.metrics

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2. 2

   Takes output from cutadapt round 1. Run to trim off the 3p™ adapters on read 2, to control for double ligation events.

   cutadapt GitHub

   $ Bash example

   cutadapt -f fastq --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.metrics

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3. 3

   Takes output from cutadapt round 2. Maps to human specific version of RepBase used to remove repetitive elements, helps control for spurious artifacts from rRNA (& other) repetitive reads.

   STAR GitHub

   $ Bash example

   STAR --runMode alignReads --runThreadN 16 --genomeDir /path/to/RepBase_human_database_file --genomeLoad LoadAndRemove --readFilesIn /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz --outSAMunmapped Within --outFilterMultimapNmax 30 --outFilterMultimapScoreRange 1 --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam --outSAMattributes All --readFilesCommand zcat --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam

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4. 4

   Takes output from STAR rmRep. Maps unique reads to the human genome.

   STAR GitHub

   $ Bash example

   STAR --runMode alignReads --runThreadN 16 --genomeDir /path/to/STAR_database_file --genomeLoad LoadAndRemove --readFilesIn /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate1 /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate2 --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam --outSAMattributes All --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam

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5. 5

   Takes output from STAR genome mapping. Custom random-mer-aware script for PCR duplicate removal.

   barcode_collapse_pe.py (inferred by models/gemini-2.5-flash) GitHub

   $ Bash example

   barcode_collapse_pe.py --bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam --out_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.bam --metrics_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.metrics

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6. 6

   Sorts resulting bam file for use downstream.

   samtools (inferred by models/gemini-2.5-flash) GitHub

   $ Bash example

   # Install samtools (if not already installed)
   # conda install -c bioconda samtools
   
   # Sort the BAM file
   samtools sort -o sorted_reads.bam input_reads.bam

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7. 7

   Command: samtools index /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam.bai

   samtools

   $ Bash example

   samtools index /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam.bai

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8. 8

   Takes inputs from multiple final bam files.

   samtools GitHub

   $ Bash example

   samtools merge /full/path/to/files/CombinedID.merged.bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.D08.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam

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9. 9

   Command: samtools index /full/path/to/files/CombinedID.merged.bam /full/path/to/files/CombinedID.merged.bam.bai

   samtools GitHub

   $ Bash example

   samtools index /full/path/to/files/CombinedID.merged.bam /full/path/to/files/CombinedID.merged.bam.bai

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10. 10

    Only outputs the second read in each pair for use with single stranded peak caller.

    samtools GitHub

    $ Bash example

    samtools view -hb -f 128 /full/path/to/files/CombinedID.merged.bam > /full/path/to/files/CombinedID.merged.r2.bam

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11. 11

    Calls peaks on those files.

    CLIPper GitHub

    $ Bash example

    clipper -b /full/path/to/files/CombinedID.merged.r2.bam -s hg19 -o /full/path/to/files/CombinedID.merged.r2.peaks.bed --bonferroni --superlocal --threshold-method binomial --save-pickle

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Tools Used