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Supplemental_Code.Rmd
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Supplemental_Code.Rmd
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---
title: Supplemental Information for 'Pioneer factor GAF cooperates with PBAP and NURF
to regulate transcription'
author:
- Julius Judd^[Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14835, USA]
- Fabiana M. Duarte\*^[Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA]
- John T. Lis\*^[Correspondance to [email protected]]
output:
pdf_document: default
html_document:
pdf_print: paged
---
\newpage
# Table of Contents
1. Supplementary Code 1. PRO-seq alignment pipeline
2. Supplementary Code 2. ATAC-seq alignment pipeline
3. Supplementary Code 3. 3' RNA-seq alignment pipeline
4. Supplementary Code 4. CUT&RUN alignment pipeline
5. Supplementary Code 5. ChIP-seq realignment pipeline
6. Supplementary Code 6. Data Analysis (R Scripts)
7. Supplementary Code 7. Session Info
This file contains all code used to analyze data presented in 'Pioneer factor GAF cooperates with PBAP and NURF to regulate transcription'.
The goal of this document is reproducibility: This is the exact code used to generate the figures in the final version of the paper, and any user should be able to replicate our analyses starting from raw data (GSE149339 or PRJNA627972).
\newpage
# Supplementary Code 1. PRO-seq alignment pipeline
This pipeline can be found here:
[http://github.com/jaj256/PROseq_alignment.sh](http://github.com/jaj256/PROseq_alignment.sh)
Analysis in this paper was performed using commit [55a08db](https://github.com/JAJ256/PROseq_alignment.sh/tree/55a08db08a1beb2582d6deec516d4684c0c9ca02)
```{bash, eval = F, echo = T}
#!/bin/bash
###########################################################
# Tue Mar 5 14:04:36 EST 2019 #
# This is a pipeline script for handling paired end #
# PRO-seq data with UMIs on both ends of the read. #
# Run this script in a directory that has one folder #
# named "fastq" which contains the data. #
# Fastq files must have identical names other than #
# ending in _R1.fastq and _R2.fastq. #
###########################################################
## Parameters
THREADS=50 # Threads to use for multithreaded applications
UMI_LEN=6 # Length of UMI in basepairs
## UMI Flags (set to Y or N as appropriate)
FIVEP_UMI="Y" # Is there a UMI on the 5' end of the read?
THREEP_UMI="Y" # Is there a UMI on the 3' end of the read?
## Adaptor sequences to clip. Default = Tru-Seq small RNA
ADAPTOR_1="TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC"
ADAPTOR_2="GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT"
## Genomes. Fill in paths.
GENOME_EXP="/home/jaj256/genome/dm6/dm6Hsp70AaOnly"
GENOME_SPIKE="/home/jaj256/genome/dm6hg38/dm6hg38" ## USE REPEAT MASKED VERSION!!
SPIKE_PREFIX="hg38" ## This is the prefix you've used on your spike in chromosomes
RDNA="/home/jaj256/genome/dm3hg38/dm3hg38rDNA"
## Mapq value for filtering multimappers
MAPQ=10
###############################################################
# PIPELINE #
###############################################################
# Unzipping if needed
echo "unzipping..."
for FILE in fastq/*
do
if [[ "$FILE" == *.gz ]]
then
gunzip $FILE &
fi
done
wait
# Removing extra info from filenames.
# This is general and works with files from Cornell BRC.
# If filenames are formatted differently, does nothing.
echo "renaming if needed..."
for FILE in $(ls fastq/)
do
NEW=fastq/"$(echo "$FILE" |
sed 's/^[0-9]\+_[0-9]\+_[0-9]\+_[0-9A-Z]\+_//' |
sed 's/_[ATCG]\{6,8\}_/_/')"
if [ ! -s "$NEW" ]
then
mv fastq/"$FILE" "$NEW"
fi
done
mkdir -p logs
mkdir -p logs/fastqc
# Running fastqc on files
echo "running fastqc if needed..."
for FILE in fastq/*.fastq
do
if [ ! -s logs/fastqc/"$(basename ${FILE/.fastq/_fastqc.zip})" ]
then
fastqc "$FILE" -o logs/fastqc --quiet &
fi
done
wait
mkdir -p trimmedFastq
# Autodetecting paired end files
echo "detecting paired end files..."
NUM=$(ls fastq | wc -l)
NUM_REDUCED=$(ls fastq | sed 's/_R.*//' | uniq | wc -l)
if [[ $NUM == $NUM_REDUCED ]]
then
PAIRED="N"
echo "detected ""$NUM"" single end fastq files. exiting..."
exit
else
PAIRED="Y"
echo "detected ""$NUM_REDUCED"" paired end fastq files"
fi
# Trimming adapters and filtering rRNA reads
# Four logical branches for 5' and 3' UMI, 5' only, 3' only, and no UMI
echo "trimming adapters and filtering rDNA reads..."
mkdir -p logs/fastp
mkdir -p logs/rRNA
mkdir -p trimmedFastq
if [[ $PAIRED == "Y" ]]
then
# Branches for either 3' UMI or both UMIs
if [[ $THREEP_UMI == "Y" ]]
then
# Branch for both UMIs
if [[ $FIVEP_UMI == "Y" ]]
then
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s trimmedFastq/${PAIR}_R1.fastq ]
then
echo "trimming adapters and filtering rRNA reads for "${PAIR}
(fastp \
-i fastq/${PAIR}_R1.fastq \
-I fastq/${PAIR}_R2.fastq \
--adapter_sequence $ADAPTOR_1 \
--adapter_sequence_r2 $ADAPTOR_2 \
--umi \
--stdout \
--umi_loc=per_read \
--umi_len=${UMI_LEN} \
--html logs/fastp/${PAIR}_fastp.html \
-w $(echo ${THREADS}/3 | bc)\
-c \
--overlap_len_require 15 2> logs/fastp/${PAIR}_fastp.log) |
(bowtie2 \
--fast-local \
--un-conc trimmedFastq/${PAIR}.fastq \
--interleaved - \
-x ${RDNA} \
--threads $(echo ${THREADS}/3*2 | bc)
2> logs/rRNA/${PAIR}_rRNA_bowtie.log) > /dev/null
fi
done
# Branch for just 3' UMI
else
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s trimmedFastq/${PAIR}_R1.fastq ]
then
echo "trimming adapters and filtering rRNA reads for "${PAIR}
(fastp \
-i fastq/${PAIR}_R1.fastq \
-I fastq/${PAIR}_R2.fastq \
--adapter_sequence $ADAPTOR_1 \
--adapter_sequence_r2 $ADAPTOR_2 \
--umi \
--stdout \
--umi_loc=read1 \
--umi_len=${UMI_LEN} \
--html logs/fastp/${PAIR}_fastp.html \
-w $(echo ${THREADS}/3 | bc) \
-c \
--overlap_len_require 15 2> logs/fastp/${PAIR}_fastp.log) |
(bowtie2 \
--fast-local \
--un-conc trimmedFastq/${PAIR}.fastq \
--interleaved - \
-x ${RDNA} \
--threads $(echo ${THREADS}/3*2 | bc)
2> logs/rRNA/${PAIR}_rRNA_bowtie.log) > /dev/null
fi
done
fi
# Branch for only 5' UMI or no UMIs
else
# Branch for only 5' UMI
if [[ $FIVEP_UMI == "Y" ]]
then
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s trimmedFastq/${PAIR}_R1.fastq ]
then
echo "trimming adapters and filtering rRNA reads for "${PAIR}
(fastp \
-i fastq/${PAIR}_R1.fastq \
-I fastq/${PAIR}_R2.fastq \
--adapter_sequence $ADAPTOR_1 \
--adapter_sequence_r2 $ADAPTOR_2 \
--umi \
--stdout \
--umi_loc=read2 \
--umi_len=${UMI_LEN} \
--html logs/fastp/${PAIR}_fastp.html \
-w $(echo ${THREADS}/3 | bc) \
-c \
--overlap_len_require 15 2> logs/fastp/${PAIR}_fastp.log) |
(bowtie2 \
--fast-local \
--un-conc trimmedFastq/${PAIR}.fastq \
--interleaved - \
-x ${RDNA} \
--threads $(echo ${THREADS}/3*2 | bc)
2> logs/rRNA/${PAIR}_rRNA_bowtie.log) > /dev/null
fi
done
# Branch for no UMI
else
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s trimmedFastq/${PAIR}_R1.fastq ]
then
echo "trimming adapters and filtering rRNA reads for "${PAIR}
(fastp \
-i fastq/${PAIR}_R1.fastq \
-I fastq/${PAIR}_R2.fastq \
--adapter_sequence $ADAPTOR_1 \
--adapter_sequence_r2 $ADAPTOR_2 \
--stdout \
--html logs/fastp/${PAIR}_fastp.html \
-w $(echo ${THREADS}/3 | bc) \
-c \
--overlap_len_require 15 2> logs/fastp/${PAIR}_fastp.log) |
(bowtie2 \
--fast-local \
--un-conc trimmedFastq/${PAIR}.fastq \
--interleaved - \
-x ${RDNA} \
--threads $(echo ${THREADS}/3*2 | bc)
2> logs/rRNA/${PAIR}_rRNA_bowtie.log) > /dev/null
fi
done
fi
fi
fi
# Cleaning up filenames in trimmedFastq (bowtie automatically names PE --un output)
for FILE in trimmedFastq/*1.fastq
do
if [ ! -s ${FILE/.1.fastq/_R1.fastq} ]
then
mv "$FILE" ${FILE/.1.fastq/_R1.fastq}
fi
done
for FILE in trimmedFastq/*2.fastq
do
if [ ! -s ${FILE/.2.fastq/_R2.fastq} ]
then
mv "$FILE" ${FILE/.2.fastq/_R2.fastq}
fi
done
# Aligning to spike in genome to get normalization factors
mkdir -p spikeBAM
mkdir -p logs/spikeAlign
if [[ "$PAIRED" == "Y" ]]
then
for PAIR in $(ls trimmedFastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s "spikeBAM/${PAIR}_hg38.BAM" ]
then
echo "aligning ${PAIR} to spike in genome"
(bowtie2 \
--local \
--very-sensitive-local \
--threads $(echo ${THREADS}/3*2 | bc) \
--no-unal \
--no-mixed \
--no-discordant \
-x "$GENOME_SPIKE" \
-1 "trimmedFastq/${PAIR}_R1.fastq" \
-2 "trimmedFastq/${PAIR}_R2.fastq" \
2> logs/spikeAlign/${PAIR}_spikeAlign.log) |
samtools view -hS -f 2 -q ${MAPQ} |
perl -n -e 'print $_ if (/^\@/ || /'${SPIKE_PREFIX}'/ ) ' |
samtools view -b |
samtools sort -@ $(echo ${THREADS}/3 | bc) -o spikeBAM/${PAIR}.BAM
samtools index spikeBAM/${PAIR}.BAM
fi
done
fi
# Aligning to experimental genome
mkdir -p BAM
mkdir -p logs/align
if [[ "$PAIRED" == "Y" ]]
then
for PAIR in $(ls trimmedFastq | sed 's/_R[1-2].*//' | uniq )
do
if [ ! -s "BAM/${PAIR}.BAM" ]
then
echo "aligning ${PAIR} to experimental genome"
(bowtie2 \
--local \
--sensitive-local \
--threads $(echo ${THREADS}/3*2 | bc) \
-x "$GENOME_EXP" \
-1 "trimmedFastq/${PAIR}_R1.fastq" \
-2 "trimmedFastq/${PAIR}_R2.fastq" \
2> logs/align/${PAIR}_align.log) |
samtools view -bS -f 2 -q ${MAPQ} |
samtools sort -@ $(echo ${THREADS}/3 | bc) -o BAM/${PAIR}.BAM
samtools index BAM/${PAIR}.BAM
fi
done
fi
# Deduplicating with UMIs (experimental BAM)
mkdir -p BAMdeDuped
mkdir -p logs/deDup
for FILE in BAM/*.BAM
do
if [ ! -s "BAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)" ]
then
(umi_tools dedup \
-I "$FILE" \
--umi-separator=":" \
--paired \
-S "BAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)" \
)> "logs/deDup/$(basename ${FILE%.BAM}_deDup.log)" &&
samtools index "BAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)"
fi
done
# Deduplicating with UMIs (Spike-In BAM)
mkdir -p spikeBAMdeDuped
mkdir -p logs/spikedeDup
for FILE in spikeBAM/*.BAM
do
if [ ! -s "spikeBAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)" ]
then
(
umi_tools dedup \
-I "$FILE" \
--paired \
--umi-separator=":" \
-S "spikeBAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)" \
)> "logs/spikedeDup/$(basename ${FILE%.BAM}_deDup.log)" &&
samtools index "spikeBAMdeDuped/$(basename ${FILE%.BAM}_deDuped.BAM)"
fi
done
# Generating table of Alignment metrics
mkdir -p info
if [ ! -s info/infoTable.tsv ]
then
touch info/infoTable.tsv
echo -e Name'\t'\
RawReads'\t'\
NonDimerReads'\t'\
%dimer'\t'\
insertSize'\t'\
rRNAreads'\t'\
%rRNA'\t'\
passedFilters'\t'\
bowtieConcordant'\t'\
bowtieMulti'\t'\
bowtieUnal'\t'\
bowtieOverallMap%'\t'\
bowtieConcordant%'\t'\
bowtieMulti%'\t'\
bowtieUnal%'\t'\
uniqueMapped'\t'\
uniqueMappedNondup'\t'\
%PCRdups'\t'\
uniqueMappedSpikein'\t'\
uniqueMappedSpikeinNondup'\t'\
spikeInPCRdups% >> info/infoTable.tsv
for SAMPLE in $(ls BAM/*.BAM | sed 's/.BAM//' | sed 's/BAM\///' )
do
NAME=${SAMPLE}
RAW_READS=$(cat logs/fastp/${SAMPLE}_fastp.log |
grep "total reads:" | head -n 1 |
awk '{print $3}')
TRIMMED_READS=$(cat logs/fastp/${SAMPLE}_fastp.log |
grep "total reads:" | tail -n 1 |
awk '{print $3}')
PER_DIMER=$(echo "(1-"${TRIMMED_READS}"/"${RAW_READS}")*100" | bc -l)%
INSERT_SIZE=$(cat logs/fastp/${SAMPLE}_fastp.log |
grep "Insert size peak" |
awk '{print $8}')
PASSED_FILTERS=$(cat logs/align/${SAMPLE}_align.log |
grep "reads; of these:$" |
awk '{print $1}')
RRNA=$(echo ${TRIMMED_READS}"-"${PASSED_FILTERS} | bc )
PER_RRNA=$(echo ${RRNA}"/"${RAW_READS}"*100" | bc -l)%
B_CONC=$(cat logs/align/${SAMPLE}_align.log |
grep "aligned concordantly exactly 1 time$" |
awk '{print $1}')
B_MULTI=$(cat logs/align/${SAMPLE}_align.log |
grep "aligned concordantly >1 times$" |
awk '{print $1}')
B_UNAL=$(cat logs/align/${SAMPLE}_align.log |
grep "aligned concordantly 0 times$" |
awk '{print $1}')
B_OAP=$(cat logs/align/${SAMPLE}_align.log |
grep "overall alignment rate$" |
awk '{print $1}')
B_CONC_PER=$(echo ${B_CONC}"/"${PASSED_FILTERS}"*100" | bc -l)%
B_MULTI_PER=$(echo ${B_MULTI}"/"${PASSED_FILTERS}"*100" | bc -l)%
B_UNAL_PER=$(echo ${B_UNAL}"/"${PASSED_FILTERS}"*100" | bc -l)%
UNIQ_MAPPED=$(cat logs/deDup/${SAMPLE}_deDup.log |
grep "Input Reads:" | awk '{print $10}')
UNIQ_MAPPED_DEDUP=$(cat logs/deDup/${SAMPLE}_deDup.log |
grep "Number of reads out:" | awk '{print $8}')
PER_DUPS=$(echo "(1-"${UNIQ_MAPPED_DEDUP}"/"${UNIQ_MAPPED}")*100" | bc -l)%
UNIQ_MAPPED_SPIKE=$(cat logs/spikedeDup/${SAMPLE}_deDup.log |
grep "Input Reads:" | awk '{print $10}')
UNIQ_MAPPED_DEDUP_SPIKE=$(cat logs/spikedeDup/${SAMPLE}_deDup.log |
grep "Number of reads out:" | awk '{print $8}')
PER_DUPS_SPIKE=$(echo "(1-"${UNIQ_MAPPED_DEDUP_SPIKE}"/"${UNIQ_MAPPED_SPIKE}")*100" |
bc -l)%
echo -e $NAME'\t'\
$RAW_READS'\t'\
$TRIMMED_READS'\t'\
$PER_DIMER'\t'\
$INSERT_SIZE'\t'\
$RRNA'\t'\
$PER_RRNA'\t'\
$PASSED_FILTERS'\t'\
$B_CONC'\t'\
$B_MULTI'\t'\
$B_UNAL'\t'\
$B_OAP'\t'\
$B_CONC_PER'\t'\
$B_MULTI_PER'\t'\
$B_UNAL_PER'\t'\
$UNIQ_MAPPED'\t'\
$UNIQ_MAPPED_DEDUP'\t'\
$PER_DUPS'\t'\
$UNIQ_MAPPED_SPIKE'\t'\
$UNIQ_MAPPED_DEDUP_SPIKE'\t'\
$PER_DUPS_SPIKE >> info/infoTable.tsv
done
fi
# Making non-normalized bigWig files
mkdir -p bw
for FILE in BAMdeDuped/*.BAM
do
if [ ! -s "bw/$(basename ${FILE/.BAM/_fwd.bw})" ]
then
bamCoverage \
--bam $FILE \
--skipNonCoveredRegions \
--outFileName bw/$(basename ${FILE/.BAM/_fwd.bw}) \
--binSize 1 \
--numberOfProcessors ${THREADS} \
--normalizeUsing None \
--Offset 1 \
--samFlagInclude 82
fi
if [ ! -s "bw/$(basename ${FILE/.BAM/_rev.bw})" ]
then
bamCoverage \
--bam $FILE \
--skipNonCoveredRegions \
--outFileName bw/$(basename ${FILE/.BAM/_rev.bw}) \
--binSize 1 \
--numberOfProcessors ${THREADS} \
--normalizeUsing None \
--Offset 1 \
--samFlagInclude 98
fi
done
```
\newpage
# Supplementary Code 2. ATAC-seq alignment pipeline
```{bash, eval = F, echo = T}
#!/bin/bash
# This script is for aligning/peak calling/making bigwig signal tracks for ATAC-seq data
# Number of threads to use for applications that support multithreading
THREADS=50
# Unzipping if needed
for FILE in fastq/*
do gunzip $FILE -q &
done
# Renaming files
for FILE in $(ls fastq/)
do
NEW=fastq/"$(echo "$FILE" |
sed 's/^[0-9]\+_[0-9]\+_[0-9]\+_[0-9A-Z]\+_//' |
sed 's/_[ATCG]\{6,8\}_/_/')"
if [ ! -s "$NEW" ]
then
mv fastq/"$FILE" "$NEW"
fi
done
# Running Fastqc
mkdir -p logs
mkdir -p logs/fastqc
for FILE in fastq/*.fastq
do
if [ ! -s logs/fastqc/"$(basename ${FILE/.fastq/_fastqc.zip})" ]
then
fastqc "$FILE" -o logs/fastqc/ --quiet &
fi
done
wait
# Aligning files
mkdir -p BAM
mkdir -p logs/align
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq)
do
if [ ! -s "BAM/${PAIR}.BAM" ]
then
(bowtie2 --local --very-sensitive-local --threads ${THREADS} \
--no-unal -I 10 -X 1000 -x ~/genome/dm6/dm6Hsp70AaOnly \
-1 fastq/${PAIR}_R1.fastq \
-2 fastq/${PAIR}_R2.fastq \
2> logs/align/${PAIR}_align.log) |
samtools view -bS -q 10 -f 2 |
samtools sort -o BAM/${PAIR}.BAM
samtools index BAM/${PAIR}.BAM
fi
done
# Plotting coverage of reads < 120 bp insert (ATAC Hypersensitivity)
mkdir -p bwDHS
for FILE in BAM/*.BAM
do
bamCoverage \
--bam ${FILE} \
--outFileName bwDHS/$(basename ${FILE/.BAM/_AtacDHS.bw}) \
--binSize 1 \
--numberOfProcessors ${THREADS} \
--normalizeUsing None \
--skipNAs \
--extendReads \
--maxFragmentLength 120
done
# Plotting coverage of centers of reads 200 > insert > 130 (mononucleosomes)
mkdir -p bwMonoNucs
for FILE in repMergedBAM/*.BAM
do
if [ ! -s bwMonoNucs/$(basename ${FILE/.BAM/_AtacMonoNucs.bw}) ]
then
bamCoverage \
--bam ${FILE} \
--outFileName bwMonoNucs/$(basename ${FILE/.BAM/_AtacMonoNucs.bw}) \
--binSize 1 \
--MNase \
--numberOfProcessors ${THREADS} \
--normalizeUsing None
fi
done
# Calling peaks on all BAM files (pooling all samples)
source /programs/bin/util/setup_macs2.sh
mkdir -p peaks
macs2 callpeak \
-t BAM/*.BAM \
-f BAMPE \
-g dm \
-n "ALL_ATAC_PEAKS" \
--outdir peaks \
--call-summits
```
\newpage
# Supplementary Code 3. 3' RNA-seq alignment pipeline
```{bash, eval = F, echo = T}
#!/bin/bash
# Number of threads to use for applications that support multithreading
THREADS=50
# building STAR index
/programs/STAR/STAR \
--runThreadN ${THREADS} \
--runMode genomeGenerate \
--genomeDir /workdir/jaj256/gaf/new/RNAseq/STARindex \
--genomeFastaFiles ~/genome/dm6hg38ERCC/dm6hg38ERCC.fa \
--sjdbGTFfile ~/genome/dm6hg38ERCC/dm6hg38ERCC.gtf \
--sjdbOverhang 68
mkdir -p trimmedFastq
mkdir -p logs
mkdir -p logs/fastp
# Trimming Adapters and polyA sequences, and extracting UMIs
for FILE in fastq/*.fastq
do
fastp \
-i ${FILE} \
-o trimmedFastq/$(basename ${FILE}) \
--length_required 25 \
--adapter_fasta adapters.fa \
--umi \
--umi_loc=read1 \
--umi_len=6 \
--html logs/fastp/$(basename ${FILE/.fastq/_fastp.html}) \
-w ${THREADS} 2> logs/$(basename ${FILE/.fastq/_fastp.log})
done
# Aligning to the combined experimental/Spike-in genome
mkdir -p BAM
mkdir -p logs/STAR
for FILE in trimmedFastq/*.fastq
do
/programs/STAR/STAR \
--runThreadN ${THREADS} \
--genomeDir STARindex/ \
--readFilesIn ${FILE} \
--outFilterType BySJout \
--outFilterMultimapNmax 20 \
--alignSJoverhangMin 8 \
--alignSJDBoverhangMin 1 \
--outFilterMismatchNmax 999 \
--outFilterMismatchNoverLmax 0.1 \
--alignIntronMin 20 \
--alignIntronMax 1000000 \
--alignMatesGapMax 1000000 \
--outSAMattributes NH HI NM MD \
--outSAMtype BAM SortedByCoordinate \
--outFileNamePrefix BAM/$(basename ${FILE/.fastq/}) \
2> logs/STAR/$(basename ${FILE/.fastq/_STAR.log})
done
# Deduplicating using UMIs
mkdir -p BAMdeDuped
mkdir -p logs/deDup
for FILE in BAM/*.BAM
do
umi_tools dedup \
-I ${FILE} \
-S "BAMdeDuped/$(basename ${FILE})" \
--umi-separator=":" \
> logs/deDup/$(basename ${FILE%.BAM}_deDup.log) &
done
wait
for FILE in BAMdeDuped/*.BAM
do samtools index $FILE &
done
wait
# Splitting spike-in and experimental alignments
mkdir -p dm6BAM
mkdir -p ERCCBAM
for FILE in BAMdeDuped/*.BAM
do
samtools view -hS -q 255 -F 4 ${FILE} |
perl -n -e 'print $_ if (/^\@/ || /ERCC/) ' |
samtools view -b |
samtools sort -o ERCCBAM/$(basename ${FILE})
samtools view -hS -q 255 -F 4 ${FILE} |
perl -n -e 'print $_ if (/^\@/ || !(/hg38/ || /ERCC/)) ' |
samtools view -b |
samtools sort -o dm6BAM/$(basename ${FILE})
done
# Counting # of alignments to experimental and spike-in genomes
mkdir -p spikeCounts
touch spikeCounts/spikeCounts.tsv
echo -e sample"\t"dm6reads"\t"ERCCreads > spikeCounts/spikeCounts.tsv
for FILE in BAMdeDuped/*.BAM;
do
FILENAME=$(basename ${FILE%.BAM})
DM6=$(samtools view -c dm6BAM/${FILENAME}.BAM)
ERCC=$(samtools view -c ERCCBAM/${FILENAME}.BAM)
echo -e ${FILENAME}"\t"${DM6}"\t"${ERCC} >> spikeCounts/spikeCounts.tsv
done
# Making bw signal tracks (non-normalized)
for FILE in dm6BAM/*.BAM
do
bamCoverage \
--bam $FILE \
--skipNonCoveredRegions \
--outFileFormat bigwig \
--outFileName bdg/$(basename ${FILE/.BAM/_fwd.bw}) \
--binSize 1 \
--numberOfProcessors ${THREADS} \
--normalizeUsing None \
--Offset 1 \
--filterRNAstrand reverse
bamCoverage \
--bam $FILE \
--skipNonCoveredRegions \
--outFileFormat bigwig \
--outFileName bdg/$(basename ${FILE/.BAM/_rev.bw}) \
--binSize 1 \
--numberOfProcessors ${THREADS} \
--normalizeUsing None \
--Offset 1 \
--filterRNAstrand forward
done
```
\newpage
# Supplementary Code 4. CUT&RUN alignment pipeline
```{bash, echo = T, eval = F}
#!/bin/bash
# Number of threads to use for applications that support multithreading
THREADS=50
# Running Fastqc
mkdir -p logs
mkdir -p logs/fastqc
for FILE in fastq/*.fastq
do
if [ ! -s logs/fastqc/$(basename ${FILE/.fastq/_fastqc.zip}) ]
then
fastqc ${FILE} -q -o logs/fastqc &
fi
done
wait
# Trimming adapters, aligning, sorting, and indexing
mkdir -p BAM
mkdir -p logs/align
mkdir -p logs/fastp
for PAIR in $(ls fastq | sed 's/_R[1-2].*//' | uniq)
do
if [ ! -s BAM/${PAIR}.BAM ]
then
(fastp \
-i fastq/${PAIR}_R1.fastq \
-I fastq/${PAIR}_R2.fastq \
--adapter_sequence AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC \
--adapter_sequence_r2 \
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT \
--stdout \
--html logs/fastq/${PAIR}_fastp.log \
-w $(echo ${THREADS}/3 | bc) \
-c --overlap_len_require 15
2> logs/fastp/${PAIR}_fastp.log) |
(bowtie2 \
--very-sensitive-local \
--no-unal --no-discordant \
--interleaved - \
-x ~/genome/dm6/dm6Hsp70AaOnly \
--threads $(echo ${THREADS}/3*2 | bc)
2> logs/align/${PAIR}_align.log) |
samtools view -bS -f 2 -q 10 |
samtools sort -o BAM/${PAIR}.BAM
samtools index BAM/${PAIR}.BAM
fi
done
# Making bigWig files
mkdir -p bw
for FILE in BAM/*.BAM;
do
if [ ! -s bw/$(basename ${FILE/BAM/bw}) ]
then
bamCoverage \
-b $FILE \
-o bw/$(basename ${FILE/BAM/bw}) \
--binSize 10 \
-p ${THREADS} \
--normalizeUsing None \
--skipNonCoveredRegions \
--extendReads \
--maxFragmentLength 120
fi
done
```
\newpage
# Supplementary Code 5. ChIP-seq realignment pipeline
```{bash, echo = T, eval = F}
#!/bin/bash
# Number of threads to use for applications that support multithreading
THREADS=50
# Aligning
mkdir -p logs/align
mkdir -p BAM
for FILE in fastq/*.fastq
do
(bowtie2 \
--very-sensitive-local \
--no-unal \
-x ~/genome/dm6/dm6Hsp70AaOnly \
--threads ${THREADS} \
-U ${FILE} 2> logs/align/$(basename ${FILE%.fastq})_align.log) |
samtools view -bS -q 10 |
samtools sort -o BAM/$(basename ${FILE%.fastq}).BAM
samtools index BAM/$(basename ${FILE%.fastq}).BAM
done
# Making bigWig files
mkdir -p bw
for FILE in BAM/*.BAM
do
bamCoverage \
-b ${FILE} \
-o bw/$(basename ${FILE/.BAM/.bw}) \
--binSize 10 \
--extendReads 200 \
--normalizeUsing None \
--skipNonCoveredRegions
done
```
\newpage
# Supplementary Code 6. Data Analysis (R Scripts)
All code chunks in this section were run in RStudio.
See Supplementary Code 7 for version of all packages used.
## Loading packages
```{r, error = F, message = F, warning = F}
library(tidyverse)
library(DESeq2)
library(ggpubr)
library(viridis)
library(scales)
library(rtracklayer)
library(GenomicRanges)
library(BiocParallel)
library(BRGenomics)
library(extrafont)
library(patchwork)
library(plyr)
library(ComplexHeatmap)
library(circlize)
library(tiff)
library(eulerr)
library(gridExtra)
loadfonts()
source("/Users/julius/Google Drive/R/customPackages/browserPlotR.R")
```
## Functions
Custom R functions used throughout the remainder of the code chunks
```{r collapse = T, error = F, message = F, warning = F}
fc_corr.jj <-
function(res1,
res2,
pval = 0.01,
cols = c("#BB0021", "#3B4992", "#BBBBBB")) {
# This function takes the path to two saved DESeq2 Results files (.tsv)
# Parses them, separates them into activated and repressed classes (padj < 0.05)
# and plots a scatter of res1 l2FC by res2 l2FC
# with a glm fit (shaded CI = 95%) of each class
# significance calling is based on the pvalue of res1
res.df <- data.frame(
res1_l2FC = as.data.frame(res1)$log2FoldChange,
res1_padj = as.data.frame(res1)$padj,
res2_l2FC = as.data.frame(res2)$log2FoldChange,
res2_padj = as.data.frame(res2)$padj
)
res.df <- res.df[!is.na(res.df$res1_padj) & !is.na(res.df$res2_l2FC),]
res.df$sig <- ifelse((res.df$res1_padj < pval),
ifelse(res.df$res1_l2FC < 0,
"down",
"up"),
"ns"
)
res.df$sig <- factor(res.df$sig, levels = c("up", "down", "ns"))
num_up <- nrow(filter(res.df, sig == "up"))
num_down <- nrow(filter(res.df, sig == "down"))
max_y <- max(res.df$res2_l2FC)
min_y <- min(res.df$res2_l2FC)
max_x <- max(res.df$res1_l2FC)
min_x <- min(res.df$res1_l2FC)
p <-
ggplot(res.df,
aes(x = res1_l2FC, y = res2_l2FC)) +
geom_abline(intercept = 0, slope = 1) +
geom_point(
data = filter(res.df, sig == "ns"),
size = 0.75,
alpha = 0.75,
stroke = 0,
show.legend = F,
color = cols[3]
) +
geom_point(
data = filter(res.df, sig == "up"),
size = 0.75,
alpha = 0.5,
stroke = 0,
show.legend = F,
color = cols[1]
) +
geom_point(
data = filter(res.df, sig == "down"),
size = 1,
alpha = 0.75,
stroke = 0,
show.legend = F,
color = cols[2]
) +
geom_smooth(
data = filter(res.df, sig == "down"),
method = "glm",
level = 0.95,
size = 0.5,
color = "black",
show.legend = F
) +
geom_smooth(
data = filter(res.df, sig == "up"),
method = "glm",
level = 0.95,
size = 0.5,
color = "black",
show.legend = F
) +
xlab(paste0(deparse(substitute(res1)), "log2 FC")) +
ylab(paste0(deparse(substitute(res2)), "log2 FC")) +
ggtheme.jj() +
geom_vline(xintercept = 0, linetype = "dashed", alpha = 0.5)+
geom_hline(yintercept = 0, linetype = "dashed", alpha = 0.5)+
annotate(geom = 'text',
label = num_up,
x = max_x * 0.9,
y = min_y * 0.9,
color = cols[1]
)+
annotate(geom = 'text',
label = num_down,
x = min_x * 0.9,
y = max_y * 0.9,
color = cols[2]
)
return(p)
}
subset_DESeq.jj <- function(df,
col_data,
scale_facts,