Duplex Workflow:

Data Pulled from:

https://s3-us-west-2.amazonaws.com/human-pangenomics/submissions/5b73fa0e-658a-4248-b2b8-cd16155bc157--UCSC_GIAB_R1041_nanopore/HG002_R1041_Duplex/1_3_23_R1041_Duplex_HG002_1.fast5.tar
https://s3-us-west-2.amazonaws.com/human-pangenomics/submissions/5b73fa0e-658a-4248-b2b8-cd16155bc157--UCSC_GIAB_R1041_nanopore/HG002_R1041_Duplex/1_3_23_R1041_Duplex_HG002_2.fast5.tar
https://s3-us-west-2.amazonaws.com/human-pangenomics/submissions/5b73fa0e-658a-4248-b2b8-cd16155bc157--UCSC_GIAB_R1041_nanopore/HG002_R1041_Duplex/1_3_23_R1041_Duplex_HG002_3.fast5.tar

Basecalling:

tar -xvf <tar>

pod5 fast5 <tar_folder> <pod5_folder>

dorado-0.6.1 duplex sup,5mCG_5hmCG <pod5_folder> > <output.bam>

Filtering:

Filters out all non-duplex reads in bam file

samtools view -b -h -d dx:1 <all_reads.bam> > <duplex.bam>

Alignment:

methyl_bam=$1
output_prefix="$(basename ${methyl_bam%.bam})"

ref_fasta=$2
ref_name="$(basename -- $ref_fasta)"
ref_name="${ref_name%.*}"

temp="/private/groups/migalab/jmmenend/tmp/"

methyl_fastq="${output_prefix}.fastq"
samtools fastq -@ 64 -T MM,ML,Mm,Ml ${methyl_bam} > ${methyl_fastq}

conda activate /private/groups/migalab/jmmenend/.conda_envs/winnowmap
meryl count k=15 output merylDB_${ref_name}_k15 ${ref_fasta}
meryl print greater-than distinct=0.9998 merylDB_${ref_name}_k15 > ${ref_name}_repetitive_k15.txt
winnowmap \
	--cs \
	--MD \
	-I 16G \
	-W ${ref_name}_repetitive_k15.txt \
	-y \
	-k 15 \
	-ax map-ont \
	${ref_fasta} ${methyl_fastq} \
	> ${output_prefix}_winnowmap.sam
conda deactivate

samtools view -bh -@ 64 ${output_prefix}_winnowmap.sam | \
	samtools sort -@ 64 - > ${output_prefix}_winnowmap_sort.bam
samtools index -@ 64 ${output_prefix}_winnowmap_sort.bam

rm ${output_prefix}_winnowmap.sam
rm ${methyl_fastq}

Secphase:

docker run \
	-v ${input_dir}:${input_dir} \
	-v ${output_dir}:${output_dir} \
	-u $(id -u):$(id -g) \
	mobinasri/secphase:v0.4.3 \
	secphase --ont \
	-i ${name_sort_output} \
	-f ${hg002v1_reference} \
	--outDir ${output_dir} \
	--prefix ${output_prefix} \
	--threads ${threads}

docker run \
	-v ${input_dir}:${input_dir} \
        -v ${output_dir}:${output_dir} \
	-u  $(id -u):$(id -g) \
	mobinasri/secphase:v0.4.3 \
	correct_bam \
	-i ${name_sort_input} \
	-P "${output_dir}/${output_prefix}.out.log" \
	-o "${output_dir}/${output_prefix}.corrected.bam" \
	--primaryOnly

Analyses:

Duplex Rate Graphs:

Script, images and example log files from dorado are within duplex_rate. The script uses log info from dorado to plot duplex rate by base on the left(as documented in the duplex portion of dorado) and by read number on the right(which is contained in the log file).

QC:

Nanoplot was run on all of the duplex files and zips for these can be found in nanoplots

WIP

Modkit:

Allows for aggregate analysis on hemi-methylation patterns.

WIP 

5mC bed tracks:

Allows us to look at hemi-methylation on the per-read basis

WIP