First functioning version, no ref

.gitignore

+ref/

README.md

 WGBS pipeline
 =============
 
+
+Preparing the VM to be able to run things
+-----------------------------------------
+
+```bash
+sudo bash prepare_vm.sh
+```
+
+
 Running a sample
 ----------------
 
+The reference genome is GRCh38 no alt (from ENCODE) to which I added the Lambda
+phage DNA to capture methylation spike-ins. It's included in the repository.
+Also included are the indices that are built using the
+"bismark_genome_preparation" utility, so no need to re-run that.
+
 ```bash
-bash pipeline_1.sh
+bash wgbs-pipeline.sh /folder/with/fastqfiles/sample_id.R1.fastq.gz /folder/with/fastqfiles/sample_id.R2.fastq.gz
 ```

apps/ext_chr_srt.py

+import sys
+
+if len(sys.argv) != 2:
+    sys.stderr.write("USAGE: ext_chr_srt.py CHR_NAME\n    CHR_NAME: chromosome name to extract (e.g. \"chrM\")\n")
+    sys.exit(1)
+
+chromosome = sys.argv[1]
+
+for line in sys.stdin:
+    if line.startswith("Bismark"):
+        sys.stdout.write(line)
+        continue
+    if line.split("\t")[2] == chromosome:
+        sys.stdout.write(line)

apps/fetch_mt_and_mate.py

+#!/usr/bin/env python3
+
+import pysam
+import sys
+
+def read_pair_generator(bam):
+    read_pair = [None,None]
+    for read in bam:
+        if not read.is_proper_pair or read.is_secondary or read.is_supplementary:
+            continue
+        if read.is_read1:
+            read_pair[0] = read
+            read_pair[1] = None
+        else:
+            read_pair[1] = read
+            if read_pair[0] is not None and read_pair[1] is not None:
+                if read_pair[0].reference_name == 'MT' or read_pair[1].reference_name == 'MT':
+                    if read_pair[0].query_name == read_pair[1].query_name:
+                        yield read_pair[0], read_pair[1]
+                    else:
+                        sys.stderr.write("Pairs not paired (or file not sorted by name)")
+                        sys.stderr.write(read_pair[0].query_name + "\n")
+                        sys.stderr.write(read_pair[1].query_name + "\n")
+                        sys.exit("Pairs not paired (or file not sorted by name)")
+            read_pair[0] = None
+            read_pair[1] = None
+
+if (len(sys.argv)<2):
+    sys.exit("Input bam file required!")
+bam = pysam.AlignmentFile(sys.argv[1], 'rb')
+outfile = pysam.AlignmentFile("-", 'w', template=bam)
+for read1, read2 in read_pair_generator(bam):
+    outfile.write(read1)
+    outfile.write(read2)
+outfile.close()
+

apps/methylation_proportion.py

+import sys
+
+total = 0
+methylated = 0
+for line in sys.stdin:
+    if line.startswith("Bismark"):
+        continue
+    if line.split("\t")[1] == '+': # Methylated
+        methylated += 1
+    total += 1
+
+sys.stdout.write("Methylation proportion: {:.2f}% ({}/{})\n".format(100*(methylated/total),methylated,total))
+        

pipeline_1.sh

-#!/bin/bash
-
-# exit when any command fails
-set -e
-trap 'last_command=$current_command; current_command=$BASH_COMMAND' DEBUG
-trap 'echo "\"${last_command}\" command filed with exit code $?"' EXIT
-
-if [ "$1" == "" ]
-then
-    echo "Provide ID!!"
-    exit 1
-fi
-
-wd="/data/bs"
-id=$1
-ref_dir="/data/ref"
-fastq_dir="/data/bs/trimmed_fq"
-
-# Programs
-bismark="/usr/local/bin/bismark-0.22.3"
-deduplicate_bismark="/usr/local/bin/deduplicate_bismark-0.22.3"
-bismark_meth_extract="/usr/local/bin/bismark_methylation_extractor-0.22.3"
-bowtie2="/usr/local/bin/bowtie2-2.4.41"
-bowtie_path="/opt/bowtie2-2.4.41/"
-samtools="/usr/local/bin/samtools-1.10"
-samtools_path="/opt/samtools-1.10"
-fetch_mt_reads="/data/bin/fetch_mt_and_mate/fetch_mt_and_mate.py"
-#samviewwithmate="/data/bin/jvarkit/dist/samviewwithmate.jar"
-#methylKit_extract="~/scripts/extractMethylation.R"
-
-# References
-b37_ref=${ref_dir}/b37_decoy/
-#nuc_ref=${ref_dir}/b37_decoy_no_mt/
-mt_ref=${ref_dir}/b37_decoy_only_mt/
-
-# Output folders
-bamfiles_1=${wd}/bamfiles_1
-mkdir -p ${bamfiles_1}
-meth_1=${wd}/meth_1
-mkdir -p ${meth_1}
-tmp_dir=${wd}/tmp_1
-mkdir -p ${tmp_dir}
-log_dir=${wd}/logs_1
-mkdir -p ${log_dir}
-
-
-# Get fastq files
-r1_files=$( find ${fastq_dir} -name *${id}*R1.fastq.gz | paste -sd "," - )
-r2_files=$( find ${fastq_dir} -name *${id}*R2*fastq.gz | paste -sd "," - )
-
-echo "R1 FILES: ${r1_files}"
-echo "R2 FILES: ${r2_files}"
-
-
-echo "#### STRATEGY 1: Align vs the whole genome (nc + mt), dedup, and extract \
-methylation of MT"
- 
-echo "     1.1 Aligning..."
-${bismark} ${b37_ref} -1 ${r1_files} -2 ${r2_files} --un --ambiguous --multicore 12 \
-    -o ${bamfiles_1} --temp_dir ${tmp_dir} --path_to_bowtie ${bowtie_path} \
-    --samtools_path ${samtools_path} > ${log_dir}/1_1_aln.stdout 2> ${log_dir}/1_1_aln.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 1.1"
-    exit 1
-fi
-
-echo "     1.2 Merging unaligned and ambiguos reads..."
-unaligned_list_r1=""
-ambig_list_r1=""
-unaligned_list_r2=""
-ambig_list_r2=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bamfiles_1}/${uns}_unmapped_reads_1.fq.gz"
-    unaligned_list_r1="${unaligned_list_r1} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bamfiles_1}/${amb}_ambiguous_reads_1.fq.gz"
-    ambig_list_r1="${ambig_list_r1} ${amb}"
-done
-IFS=',' read -r -a array <<< "${r2_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bamfiles_1}/${uns}_unmapped_reads_2.fq.gz"
-    unaligned_list_r2="${unaligned_list_r2} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bamfiles_1}/${amb}_ambiguous_reads_2.fq.gz"
-    ambig_list_r2="${ambig_list_r2} ${amb}"
-done
-
-cat ${unaligned_list_r1} > ${bamfiles_1}/${id}_unmapped.R1.fastq.gz
-cat ${ambig_list_r1} > ${bamfiles_1}/${id}_ambiguous.R1.fastq.gz
-cat ${unaligned_list_r2} > ${bamfiles_1}/${id}_unmapped.R2.fastq.gz
-cat ${ambig_list_r2} > ${bamfiles_1}/${id}_ambiguous.R2.fastq.gz
-
-if [ -s "${bamfiles_1}/${id}_unmapped.R1.fastq.gz" ]; then
-    rm ${unaligned_list_r1}
-fi
-if [ -s "${bamfiles_1}/${id}_unmapped.R2.fastq.gz" ]; then
-    rm ${unaligned_list_r2}
-fi
-if [ -s "${bamfiles_1}/${id}_ambiguous.R1.fastq.gz" ]; then
-    rm ${ambig_list_r1}
-fi
-if [ -s "${bamfiles_1}/${id}_ambiguous.R2.fastq.gz" ]; then
-    rm ${ambig_list_r2}
-fi
-
-
-echo "     1.3 Merging bamfiles..."
-bamfile_list=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    bamfile=$( basename ${element} )
-    bamfile="${bamfiles_1}/${bamfile%.fastq.gz}_bismark_bt2_pe.bam"
-    bamfile_list="${bamfile_list} ${bamfile}"
-done
-
-${samtools} merge -n -r -@ 64 ${bamfiles_1}/${id}_b37.bam ${bamfile_list} > \
-    ${log_dir}/1_3_merge.stdout 2> \
-    ${log_dir}/1_3_merge.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 1.3"
-    exit 1
-fi
-
-if [ -s "${bamfiles_1}/${id}_b37.bam" ]; then
-    echo "         Removing lane bams..."
-    rm ${bamfile_list}
-fi
-
-
-echo "     1.4 Dedupping alignment..."
-${deduplicate_bismark} --paired --samtools_path ${samtools_path} \
-    --output_dir ${bamfiles_1} ${bamfiles_1}/${id}_b37.bam > \
-    ${log_dir}/1_4_dedup.stdout 2> \
-    ${log_dir}/1_4_dedup.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 1.4"
-    exit 1
-fi
-
-if [ -s "${bamfiles_1}/${id}_b37.deduplicated.bam" ]; then
-    mv ${bamfiles_1}/${id}_b37.deduplicated.bam ${bamfiles_1}/${id}_b37.dedup.bam
-fi
-
-
-echo "     1.5 Extracting methylation in MT..."
-${bismark_meth_extract} -p --comprehensive --output ${meth_1} \
-    --gzip --multicore 16 --bedGraph --genome_folder ${b37_ref} \
-    --CX --samtools_path ${samtools_path} --ample_memory \
-    ${bamfiles_1}/${id}_b37.dedup.bam > \
-    ${log_dir}/1_5_meth.stdout 2> \
-    ${log_dir}/1_5_meth.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 1.5"
-    exit 1
-fi
-
-
-# echo "     1.6 Fetching MT reads (and their pairs)..."
-# python3 ${fetch_mt_reads} ${bamfiles_1}/${id}_b37.dedup.bam | \
-#     ${samtools} view -@ 16 -b -o ${bamfiles_1}/${id}_mt_reads_and_mates.bam -
-
-
-

pipeline_2.sh

-#!/bin/bash
-
-# exit when any command fails
-set -e
-trap 'last_command=$current_command; current_command=$BASH_COMMAND' DEBUG
-trap 'echo "\"${last_command}\" command filed with exit code $?"' EXIT
-
-if [ "$1" == "" ]
-then
-    echo "Provide ID!!"
-    exit 1
-fi
-
-wd="/data/bs"
-id=$1
-ref_dir="/data/ref"
-fastq_dir="/data/bs/trimmed_fq"
-
-# Programs
-bismark="/usr/local/bin/bismark-0.22.3"
-deduplicate_bismark="/usr/local/bin/deduplicate_bismark-0.22.3"
-bismark_meth_extract="/usr/local/bin/bismark_methylation_extractor-0.22.3"
-bowtie2="/usr/local/bin/bowtie2-2.4.41"
-bowtie_path="/opt/bowtie2-2.4.41/"
-samtools="/usr/local/bin/samtools-1.10"
-samtools_path="/opt/samtools-1.10"
-fetch_mt_reads="/data/bin/fetch_mt_and_mate/fetch_mt_and_mate.py"
-#samviewwithmate="/data/bin/jvarkit/dist/samviewwithmate.jar"
-#methylKit_extract="~/scripts/extractMethylation.R"
-
-# References
-#b37_ref=${ref_dir}/b37_decoy/
-#nuc_ref=${ref_dir}/b37_decoy_no_mt/
-mt_ref=${ref_dir}/b37_decoy_only_mt/
-
-# Output folders
-bam_dir=${wd}/bamfiles_2
-mkdir -p ${bam_dir}
-meth_dir=${wd}/meth_2
-mkdir -p ${meth_dir}
-tmp_dir=${wd}/tmp_2
-mkdir -p ${tmp_dir}
-log_dir=${wd}/logs_2
-mkdir -p ${log_dir}
-
-
-# Get fastq files
-r1_files=$( find ${fastq_dir} -name *${id}*R1.fastq.gz | paste -sd "," - )
-r2_files=$( find ${fastq_dir} -name *${id}*R2*fastq.gz | paste -sd "," - )
-
-echo "R1 FILES: ${r1_files}"
-echo "R2 FILES: ${r2_files}"
-
-
-echo "#### STRATEGY 2: Align vs mitochondrial genome only (mt), dedup, and extract \
-methylation"
- 
-echo "     2.1 Aligning..."
-${bismark} ${mt_ref} -1 ${r1_files} -2 ${r2_files} --un --ambiguous --multicore 12 \
-    -o ${bam_dir} --temp_dir ${tmp_dir} --path_to_bowtie ${bowtie_path} \
-    --samtools_path ${samtools_path} > ${log_dir}/2_1_aln.stdout 2> ${log_dir}/2_1_aln.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 2.1"
-    exit 1
-fi
-
-echo "     2.2 Merging unaligned and ambiguos reads..."
-unaligned_list_r1=""
-ambig_list_r1=""
-unaligned_list_r2=""
-ambig_list_r2=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bam_dir}/${uns}_unmapped_reads_1.fq.gz"
-    unaligned_list_r1="${unaligned_list_r1} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bam_dir}/${amb}_ambiguous_reads_1.fq.gz"
-    ambig_list_r1="${ambig_list_r1} ${amb}"
-done
-IFS=',' read -r -a array <<< "${r2_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bam_dir}/${uns}_unmapped_reads_2.fq.gz"
-    unaligned_list_r2="${unaligned_list_r2} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bam_dir}/${amb}_ambiguous_reads_2.fq.gz"
-    ambig_list_r2="${ambig_list_r2} ${amb}"
-done
-
-cat ${unaligned_list_r1} > ${bam_dir}/${id}_unmapped.R1.fastq.gz
-cat ${ambig_list_r1} > ${bam_dir}/${id}_ambiguous.R1.fastq.gz
-cat ${unaligned_list_r2} > ${bam_dir}/${id}_unmapped.R2.fastq.gz
-cat ${ambig_list_r2} > ${bam_dir}/${id}_ambiguous.R2.fastq.gz
-
-if [ -s "${bam_dir}/${id}_unmapped.R1.fastq.gz" ]; then
-    rm ${unaligned_list_r1}
-fi
-if [ -s "${bam_dir}/${id}_unmapped.R2.fastq.gz" ]; then
-    rm ${unaligned_list_r2}
-fi
-if [ -s "${bam_dir}/${id}_ambiguous.R1.fastq.gz" ]; then
-    rm ${ambig_list_r1}
-fi
-if [ -s "${bam_dir}/${id}_ambiguous.R2.fastq.gz" ]; then
-    rm ${ambig_list_r2}
-fi
-
-
-echo "     2.3 Merging bamfiles..."
-bamfile_list=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    bamfile=$( basename ${element} )
-    bamfile="${bam_dir}/${bamfile%.fastq.gz}_bismark_bt2_pe.bam"
-    bamfile_list="${bamfile_list} ${bamfile}"
-done
-
-${samtools} merge -n -r -@ 64 ${bam_dir}/${id}_mt.bam ${bamfile_list} > \
-    ${log_dir}/2_3_merge.stdout 2> \
-    ${log_dir}/2_3_merge.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 2.3"
-    exit 1
-fi
-
-if [ -s "${bam_dir}/${id}_mt.bam" ]; then
-    echo "         Removing lane bams..."
-    rm ${bamfile_list}
-fi
-
-
-echo "     2.4 Dedupping alignment..."
-${deduplicate_bismark} --paired --samtools_path ${samtools_path} \
-    --output_dir ${bam_dir} ${bam_dir}/${id}_mt.bam > \
-    ${log_dir}/2_4_dedup.stdout 2> \
-    ${log_dir}/2_4_dedup.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 2.4"
-    exit 1
-fi
-
-if [ -s "${bam_dir}/${id}_mt.deduplicated.bam" ]; then
-    mv ${bam_dir}/${id}_mt.deduplicated.bam ${bam_dir}/${id}_mt.dedup.bam
-fi
- 
- 
-echo "     2.5 Extracting methylation in MT..."
-${bismark_meth_extract} -p --comprehensive --output ${meth_dir} \
-    --gzip --multicore 16 --cytosine_report --genome_folder ${mt_ref} \
-    --CX --samtools_path ${samtools_path} \
-    ${bam_dir}/${id}_mt.dedup.bam > \
-    ${log_dir}/2_5_meth.stdout 2> \
-    ${log_dir}/2_5_meth.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 2.5"
-    exit 1
-fi
-
-
-echo "     2.6 Fetching MT reads (and their pairs)..."
-python3 ${fetch_mt_reads} ${bam_dir}/${id}_mt.dedup.bam | \
-    ${samtools} view -@ 16 -b -o ${bam_dir}/${id}_mt_reads_and_mates.bam -
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 2.6"
-    exit 1
-fi
-
-
-

pipeline_3.sh

-#!/bin/bash
-
-# exit when any command fails
-set -e
-trap 'last_command=$current_command; current_command=$BASH_COMMAND' DEBUG
-trap 'echo "\"${last_command}\" command filed with exit code $?"' EXIT
-
-if [ "$1" == "" ]
-then
-    echo "Provide ID!!"
-    exit 1
-fi
-
-wd="/data/bs"
-id=$1
-ref_dir="/data/ref"
-fastq_dir="/data/bs/trimmed_fq"
-
-# Programs
-bismark="/usr/local/bin/bismark-0.22.3"
-deduplicate_bismark="/usr/local/bin/deduplicate_bismark-0.22.3"
-bismark_meth_extract="/usr/local/bin/bismark_methylation_extractor-0.22.3"
-bismark_bedGraph="/usr/local/bin/bismark2bedGraph-0.22.3"
-bowtie2="/usr/local/bin/bowtie2-2.4.41"
-bowtie_path="/opt/bowtie2-2.4.41/"
-samtools="/usr/local/bin/samtools-1.10"
-samtools_path="/opt/samtools-1.10"
-fetch_mt_reads="/data/bin/fetch_mt_and_mate/fetch_mt_and_mate.py"
-ext_mt_srt="/data/bs/ext_mt_srt.py"
-#samviewwithmate="/data/bin/jvarkit/dist/samviewwithmate.jar"
-#methylKit_extract="~/scripts/extractMethylation.R"
-
-# References
-b37_ref=${ref_dir}/b37_decoy/
-nuc_ref=${ref_dir}/b37_decoy_no_mt/
-mt_ref=${ref_dir}/b37_decoy_only_mt/
-
-# Output folders
-bam_dir=${wd}/bamfiles_3
-mkdir -p ${bam_dir}
-meth_dir=${wd}/meth_3
-mkdir -p ${meth_dir}
-tmp_dir=${wd}/tmp_3
-mkdir -p ${tmp_dir}
-log_dir=${wd}/logs_3
-mkdir -p ${log_dir}
-
-
-# Get fastq files
-r1_files=$( find ${fastq_dir} -name *${id}*R1.fastq.gz | paste -sd "," - )
-r2_files=$( find ${fastq_dir} -name *${id}*R2*fastq.gz | paste -sd "," - )
-
-echo "R1 FILES: ${r1_files}"
-echo "R2 FILES: ${r2_files}"
-
-
-echo "#### STRATEGY 3: Align vs nuclear DNA, collect unmapped and align them to mt DNA \
-methylation"
- 
-echo "     3.1 Aligning vs nuclear DNA..."
-${bismark} ${nuc_ref} -1 ${r1_files} -2 ${r2_files} --un --ambiguous --multicore 12 \
-    -o ${bam_dir} --temp_dir ${tmp_dir} --path_to_bowtie ${bowtie_path} \
-    --samtools_path ${samtools_path} > ${log_dir}/3_1_aln.stdout 2> ${log_dir}/3_1_aln.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 3.1"
-    exit 1
-fi
-
-echo "     3.2 Merging unaligned and ambiguos reads..."
-unaligned_list_r1=""
-ambig_list_r1=""
-unaligned_list_r2=""
-ambig_list_r2=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bam_dir}/${uns}_unmapped_reads_1.fq.gz"
-    unaligned_list_r1="${unaligned_list_r1} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bam_dir}/${amb}_ambiguous_reads_1.fq.gz"
-    ambig_list_r1="${ambig_list_r1} ${amb}"
-done
-IFS=',' read -r -a array <<< "${r2_files}"
-for element in "${array[@]}"; do
-    uns=$( basename ${element} )
-    uns="${bam_dir}/${uns}_unmapped_reads_2.fq.gz"
-    unaligned_list_r2="${unaligned_list_r2} ${uns}"
-    amb=$( basename ${element} )
-    amb="${bam_dir}/${amb}_ambiguous_reads_2.fq.gz"
-    ambig_list_r2="${ambig_list_r2} ${amb}"
-done
-
-cat ${unaligned_list_r1} > ${bam_dir}/${id}_unmapped.R1.fastq.gz
-cat ${ambig_list_r1} > ${bam_dir}/${id}_ambiguous.R1.fastq.gz
-cat ${unaligned_list_r2} > ${bam_dir}/${id}_unmapped.R2.fastq.gz
-cat ${ambig_list_r2} > ${bam_dir}/${id}_ambiguous.R2.fastq.gz
-
-if [ -s "${bam_dir}/${id}_unmapped.R1.fastq.gz" ]; then
-    rm ${unaligned_list_r1}
-fi
-if [ -s "${bam_dir}/${id}_unmapped.R2.fastq.gz" ]; then
-    rm ${unaligned_list_r2}
-fi
-if [ -s "${bam_dir}/${id}_ambiguous.R1.fastq.gz" ]; then
-    rm ${ambig_list_r1}
-fi
-if [ -s "${bam_dir}/${id}_ambiguous.R2.fastq.gz" ]; then
-    rm ${ambig_list_r2}
-fi
-
-
-echo "     3.3 Merging bamfiles..."
-bamfile_list=""
-IFS=',' read -r -a array <<< "${r1_files}"
-for element in "${array[@]}"; do
-    bamfile=$( basename ${element} )
-    bamfile="${bam_dir}/${bamfile%.fastq.gz}_bismark_bt2_pe.bam"
-    bamfile_list="${bamfile_list} ${bamfile}"
-done
-
-${samtools} merge -n -r -@ 64 ${bam_dir}/${id}_nuc.bam ${bamfile_list} > \
-    ${log_dir}/3_3_merge.stdout 2> \
-    ${log_dir}/3_3_merge.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 3.3"
-    exit 1
-fi
-
-if [ -s "${bam_dir}/${id}_nuc.bam" ]; then
-    echo "         Removing lane bams..."
-    rm ${bamfile_list}
-fi
-
-
-## Deduplication not really needed
-## echo "     3.4 Dedupping alignment..."
-## ${deduplicate_bismark} --paired --samtools_path ${samtools_path} \
-##     --output_dir ${bam_dir} ${bam_dir}/${id}_nuc.bam > \
-##     ${log_dir}/3_4_dedup.stdout 2> \
-##     ${log_dir}/3_4_dedup.stderr
-## if [ "$?" != "0" ]; then
-##     echo "ERROR IN STEP 3.4"
-##     exit 1
-## fi
-## 
-## if [ -s "${bam_dir}/${id}_nuc.deduplicated.bam" ]; then
-##     mv ${bam_dir}/${id}_nuc.deduplicated.bam ${bam_dir}/${id}_nuc.dedup.bam
-## fi
-
-echo "     3.4 Re-aligning unmapped reads to mtDNA..."
-${bismark} ${mt_ref} -1 ${bam_dir}/${id}_unmapped.R1.fastq.gz \
-    -2 ${bam_dir}/${id}_unmapped.R2.fastq.gz --multicore 12 \
-    -o ${bam_dir} --temp_dir ${tmp_dir} --path_to_bowtie ${bowtie_path} \
-    --samtools_path ${samtools_path} > \
-    ${log_dir}/3_4_mt_map.stdout 2> \
-    ${log_dir}/3_4_mt_map.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 3.4"
-    exit 1
-fi
-
-mv ${bam_dir}/${id}_unmapped.R1_bismark_bt2_pe.bam ${bam_dir}/${id}_unmapped_to_mt.bam
-
-echo "     3.5 Re-aligning unmapped reads to nuc+mt..."
-${bismark} ${b37_ref} -1 ${bam_dir}/${id}_unmapped.R1.fastq.gz \
-    -2 ${bam_dir}/${id}_unmapped.R2.fastq.gz --multicore 12 \
-    -o ${bam_dir} --temp_dir ${tmp_dir} --path_to_bowtie ${bowtie_path} \
-    --samtools_path ${samtools_path} > \
-    ${log_dir}/3_5_b37_map.stdout 2> \
-    ${log_dir}/3_5_b37_map.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 3.4"
-    exit 1
-fi
-
-mv ${bam_dir}/${id}_unmapped.R1_bismark_bt2_pe.bam ${bam_dir}/${id}_unmapped_to_b37.bam
-
-echo "     3.6 Dedupping alignments..."
-${deduplicate_bismark} --paired --samtools_path ${samtools_path} \
-    --output_dir ${bam_dir} ${bam_dir}/${id}_unmapped_to_mt.bam > \
-    ${log_dir}/3_6_a_dedup.stdout 2> \
-    ${log_dir}/3_6_a_dedup.stderr
-if [ "$?" != "0" ]; then
-    echo "ERROR IN STEP 3.6.a"