From aa79d724688f10f77e20bd2c19118da7dffa6b1e Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Wed, 2 Aug 2023 10:17:32 -0400 Subject: [PATCH 01/10] Initial draft of Hi-C workflow --- text/hic-workflow.md | 53 ++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 53 insertions(+) create mode 100644 text/hic-workflow.md diff --git a/text/hic-workflow.md b/text/hic-workflow.md new file mode 100644 index 0000000..492972f --- /dev/null +++ b/text/hic-workflow.md @@ -0,0 +1,53 @@ +# Hi-C Pipeline + +- [Introduction](#introduction) +- [Motivation](#motivation) +- [Discussion](#discussion) +- [Specification](#specification) + +# Introduction + +This RFC lays out the specification for the Hi-C harmonization pipeline. + +# Motivation + +To provide the community access to data from Hi-C experiments performed at St. Jude Children's Research Hospital, we propose the following data harmonization pipeline. The goal of this pipeline is to provide harmonized files for Hi-C data. For this pipeline, we will make no recommendations on downstream analysis, focusing instead on harmonizing the underlying sequencing data and leaving analysis decisions to the user. + +# Discussion + +Hi-C investigates chromatin conformation. + +# Specification + +## Dependencies + +- Picard Tools + +## Reference Files + +There are no reference files for the Hi-C harmonization workflow. + + +## Workflow + +Here are the resulting steps in the Hi-C workflow. There might be slight alterations in the actual implementation, which can be found in [the St. Jude Cloud workflows repository](https://github.com/stjudecloud/workflows/blob/master/workflows/hic/hic-standard.wdl). + +1. Run `picard ValidateSam` on the incoming BAM to ensure that it is well-formed enough to convert back to FastQ. + + ```bash + picard ValidateSamFile \ + I=$INPUT_BAM \ # Input BAM. + IGNORE=INVALID_PLATFORM_VALUE \ # Validations to ignore. + IGNORE=MISSING_PLATFORM_VALUE + ``` + +2. Run Picard `RevertSam` on each of the BAMs generated in the previous step. + + ```bash + picard RevertSam \ + INPUT=$INPUT_BAM \ + OUTPUT=$REVERTED_BAM \ + REMOVE_ALIGNMENT_INFORMATION=true \ + REMOVE_DUPLICATE_INFORMATION=true \ + VALIDATION_STRINGENCY=SILENT + ``` \ No newline at end of file From 2a89edea60ecd8b283dd0edb0d13159dcb59df9b Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Wed, 2 Aug 2023 10:19:34 -0400 Subject: [PATCH 02/10] Minor update --- text/hic-workflow.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index 492972f..c1e9dfb 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -32,7 +32,7 @@ There are no reference files for the Hi-C harmonization workflow. Here are the resulting steps in the Hi-C workflow. There might be slight alterations in the actual implementation, which can be found in [the St. Jude Cloud workflows repository](https://github.com/stjudecloud/workflows/blob/master/workflows/hic/hic-standard.wdl). -1. Run `picard ValidateSam` on the incoming BAM to ensure that it is well-formed enough to convert back to FastQ. +1. Run `picard ValidateSam` on the incoming BAM to ensure that it is well-formed enough to strip alignment information. ```bash picard ValidateSamFile \ @@ -41,7 +41,7 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat IGNORE=MISSING_PLATFORM_VALUE ``` -2. Run Picard `RevertSam` on each of the BAMs generated in the previous step. +2. Run Picard `RevertSam` on the validated BAM file. ```bash picard RevertSam \ From 27197b3d8a50b9f8a4f0288fa8fdf9517c0cdaf3 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Tue, 22 Aug 2023 14:55:37 -0400 Subject: [PATCH 03/10] Set sort order explicitly --- text/hic-workflow.md | 11 ++++++----- 1 file changed, 6 insertions(+), 5 deletions(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index c1e9dfb..73d3c85 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -45,9 +45,10 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat ```bash picard RevertSam \ - INPUT=$INPUT_BAM \ - OUTPUT=$REVERTED_BAM \ - REMOVE_ALIGNMENT_INFORMATION=true \ - REMOVE_DUPLICATE_INFORMATION=true \ - VALIDATION_STRINGENCY=SILENT + INPUT=$INPUT_BAM \ # Input BMA to revert + OUTPUT=$REVERTED_BAM \ # Output unaligned BAM name + REMOVE_ALIGNMENT_INFORMATION=true \ # Remove alignments + REMOVE_DUPLICATE_INFORMATION=true \ # Remove duplicate flags + VALIDATION_STRINGENCY=SILENT \ # Ignore some validation warnings + SORT_ORDER=queryname # Sort by queryname ``` \ No newline at end of file From 1504d33c7c3bb708caf967c5fb24063a994e1440 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Tue, 22 Aug 2023 15:45:05 -0400 Subject: [PATCH 04/10] Add additional detail and images to the description of Hi-C --- .../hic-workflow/Hi-C_Workflow_hi-def.png | Bin 0 -> 31960 bytes ...nfirmation-capture-wikipedia-60percent.png | Bin 0 -> 435385 bytes text/hic-workflow.md | 21 +++++++++++++++--- 3 files changed, 18 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Date: Tue, 29 Aug 2023 09:37:32 -0400 Subject: [PATCH 05/10] Further information --- text/hic-workflow.md | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index ce6bece..4b5d52e 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -10,7 +10,7 @@ This RFC lays out the specification for the Hi-C harmonization pipeline. Hi-C is a high-throughput epigenetic technique to investigate chromatin conformation. -As an epigenetic technique, the goals of a typical Hi-C experiment differ from those typical of experiments using genomic or transcriptomic sequencing methods (e.g. Whole-Genome, Whole-Exome, and RNA-Seq) that are more commonly curated by St. Jude Cloud. Epigenetics can be defined as "the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence" ([Dupont, 2009](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791696)). The latter part of this definition (_"that do not entail a change in DNA sequence"_) is vital to understanding how ATAC-Seq (and other epigentic techniques) treat sequenced data. Often, the reads are merely useful for identifying the location of epigenetic events. +As an epigenetic technique, the goals of a typical Hi-C experiment differ from those typical of experiments using genomic or transcriptomic sequencing methods (e.g. Whole-Genome, Whole-Exome, and RNA-Seq) that are more commonly curated by St. Jude Cloud. Epigenetics can be defined as "the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence" ([Dupont, 2009](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791696)). The latter part of this definition (_"that do not entail a change in DNA sequence"_) is vital to understanding how Hi-C (and other epigentic techniques) treat sequenced data. Often, the reads are merely useful for identifying the location of epigenetic events. # Motivation @@ -27,6 +27,9 @@ The Hi-C method measures the frequency at which two fragments physically associa ![Hi-C schematic](../resources/hic-workflow/Hi-C_Workflow_hi-def.png) [source](https://www.activemotif.com/catalog/1317/hi-c-service) +Though Hi-C experiments utilize paired-end sequencing, the reads can not be mapped using the paired-end mode of aligners. Since the two reads in the pair come from different fragments, the insert size of the pair is highly variable and this violates the assumptions that most paired-end aligners make. Therefore reads are often mapped as independent single-end reads. The goal of finding the ligation junctions in the Hi-C experiment have led to most projects using an iterative mapping strategy. Often starting with a truncated read and searching for a unique mapping. Non-uniquely mapped reads are then extended and an additional alignment run is performed. This can be done iteratively until the reads are either uniquely mapped or no unique mapping is found for each read. Only reads with both ends uniquely mapped are retained for analysis. + +At Jinghui's direction, this workflow will provide Hi-C data in a raw format. We will opt to provide this in unaligned BAM format. # Specification ## Dependencies From 6b0751e294d1d01e7df30a832ccf362208772975 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Tue, 29 Aug 2023 15:34:14 -0400 Subject: [PATCH 06/10] Add final validation step --- text/hic-workflow.md | 9 +++++++++ 1 file changed, 9 insertions(+) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index 4b5d52e..d3ee987 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -66,6 +66,15 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat SORT_ORDER=queryname # Sort by queryname ``` +3. Run `picard ValidateSam` on the reverted BAM to ensure that it is well-formed. + + ```bash + picard ValidateSamFile \ + I=$REVERTED_BAM \ # Input BAM. + IGNORE=INVALID_PLATFORM_VALUE \ # Validations to ignore. + IGNORE=MISSING_PLATFORM_VALUE + ``` + # Further Reading - [The Hitchhiker's Guide to Hi-C Analysis: Practical guidelines](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347522/) From acb94d72b6d8081916ca2925837df0c890ce06bf Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Thu, 4 Apr 2024 12:59:34 -0400 Subject: [PATCH 07/10] Rework Hi-C workflow proposal --- text/hic-workflow.md | 81 ++++++++++++++++++++++++++++++++++++-------- 1 file changed, 67 insertions(+), 14 deletions(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index d3ee987..4c8c9d4 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -30,16 +30,37 @@ The Hi-C method measures the frequency at which two fragments physically associa Though Hi-C experiments utilize paired-end sequencing, the reads can not be mapped using the paired-end mode of aligners. Since the two reads in the pair come from different fragments, the insert size of the pair is highly variable and this violates the assumptions that most paired-end aligners make. Therefore reads are often mapped as independent single-end reads. The goal of finding the ligation junctions in the Hi-C experiment have led to most projects using an iterative mapping strategy. Often starting with a truncated read and searching for a unique mapping. Non-uniquely mapped reads are then extended and an additional alignment run is performed. This can be done iteratively until the reads are either uniquely mapped or no unique mapping is found for each read. Only reads with both ends uniquely mapped are retained for analysis. At Jinghui's direction, this workflow will provide Hi-C data in a raw format. We will opt to provide this in unaligned BAM format. + # Specification ## Dependencies - Picard Tools +- samtools +- bwa +- pairix +- juicer ## Reference Files -There are no reference files for the Hi-C harmonization workflow. +The following reference files are used as the basis of the Hi-C Workflow: + +- Similarly to all analysis pipelines in St. Jude Cloud, we use the `GRCh38_no_alt` analysis set for our reference genome. You can get a copy of the file [here](https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz). Additionally, you can get the file by running the following commands: + + ```bash + wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz -O GRCh38_no_alt.fa.gz + gunzip GRCh38_no_alt.fa.gz + + echo "a6da8681616c05eb542f1d91606a7b2f GRCh38_no_alt.fa" > GRCh38_no_alt.fa.md5 + md5sum -c GRCh38_no_alt.fa.md5 + # > GRCh38_no_alt.fa: OK + ``` +- The following command is used to prepare the BWA index file: + + ```bash + bwa index GRCh38_no_alt.fa + ``` ## Workflow @@ -54,27 +75,59 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat IGNORE=MISSING_PLATFORM_VALUE ``` -2. Run Picard `RevertSam` on the validated BAM file. +2. Run samtools `collate` on the validated BAM file. ```bash - picard RevertSam \ - INPUT=$INPUT_BAM \ # Input BMA to revert - OUTPUT=$REVERTED_BAM \ # Output unaligned BAM name - REMOVE_ALIGNMENT_INFORMATION=true \ # Remove alignments - REMOVE_DUPLICATE_INFORMATION=true \ # Remove duplicate flags - VALIDATION_STRINGENCY=SILENT \ # Ignore some validation warnings - SORT_ORDER=queryname # Sort by queryname + samtools collate \ + -o $COLLATED_BAM \ + $INPUT_BAM ``` -3. Run `picard ValidateSam` on the reverted BAM to ensure that it is well-formed. +3. Run Samtools `fastq` on the collated BAM file. ```bash - picard ValidateSamFile \ - I=$REVERTED_BAM \ # Input BAM. - IGNORE=INVALID_PLATFORM_VALUE \ # Validations to ignore. - IGNORE=MISSING_PLATFORM_VALUE + samtools fastq \ + -1 $READ_ONE_GZ \ # Fastq with read 1s + -2 $READ_TWO_GZ \ # Fastq with read 2s + $COLLATED_BAM # Input BMA to convert to fastq + ``` +4. Run picard `FastqToSam` on fastq files to create unaligned BAM file. + ```bash + picard FastqToSam \ + FASTQ=$READ_ONE_GZ \ # Fastq with read 1s + FASTQ2=$READ_TWO_GZ \ # Fastq with read 2s + OUTPUT=$UNALIGNED_BAM # Output unaligned BAM file + ``` + +5. Run bwa `mem` on fastq files to create aligned BAM file. + + ```bash + bwa mem \ + $BWA_DB \ # BWA reference database + $READ_ONE_GZ \ # Fastq with read 1s + $READ_TWO_GZ \ # Fastq with read 2s + | samtools view -hb \ # Convert output SAM to BAM + > $ALIGNED_BAM + ``` + +6. Run pairix `bam2pairs` on aligned BAM file. + + ```bash + bam2pairs \ + $ALIGNED_BAM \ # BAM file from bwa mem + $PAIRS # Output mapped read pairs + ``` + +7. Run Juicer `pre` to generate .hic file. + + ```bash + juicer_tools pre \ + $PAIRS \ # Reads with both mates mapped + $HIC \ # hic file with multiple resolutions + $GENOME_ID # Predefined genome matching alignment + ``` # Further Reading - [The Hitchhiker's Guide to Hi-C Analysis: Practical guidelines](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347522/) From 7015b205661be2936578c4d34b23497918c67529 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Thu, 4 Apr 2024 13:51:45 -0400 Subject: [PATCH 08/10] Add restriction sites --- text/hic-workflow.md | 7 ++++--- 1 file changed, 4 insertions(+), 3 deletions(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index 4c8c9d4..83d89fc 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -124,9 +124,10 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat ```bash juicer_tools pre \ - $PAIRS \ # Reads with both mates mapped - $HIC \ # hic file with multiple resolutions - $GENOME_ID # Predefined genome matching alignment + -f $RESTRICTION_SITES \ # List of restriction sites, if available + $PAIRS \ # Reads with both mates mapped + $HIC \ # hic file with multiple resolutions + $GENOME_ID # Predefined genome matching alignment ``` # Further Reading From 60c673b92126260277c27484c40a13cdbbcc33e4 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Thu, 5 Sep 2024 11:49:43 -0400 Subject: [PATCH 09/10] refactor: Update Hi-C RFC to reflect HiC-Pro usage. --- text/hic-workflow.md | 626 ++++++++++++++++++++++++++++++++++++++++--- 1 file changed, 588 insertions(+), 38 deletions(-) diff --git a/text/hic-workflow.md b/text/hic-workflow.md index 83d89fc..4baf509 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -29,17 +29,16 @@ The Hi-C method measures the frequency at which two fragments physically associa Though Hi-C experiments utilize paired-end sequencing, the reads can not be mapped using the paired-end mode of aligners. Since the two reads in the pair come from different fragments, the insert size of the pair is highly variable and this violates the assumptions that most paired-end aligners make. Therefore reads are often mapped as independent single-end reads. The goal of finding the ligation junctions in the Hi-C experiment have led to most projects using an iterative mapping strategy. Often starting with a truncated read and searching for a unique mapping. Non-uniquely mapped reads are then extended and an additional alignment run is performed. This can be done iteratively until the reads are either uniquely mapped or no unique mapping is found for each read. Only reads with both ends uniquely mapped are retained for analysis. -At Jinghui's direction, this workflow will provide Hi-C data in a raw format. We will opt to provide this in unaligned BAM format. - # Specification ## Dependencies -- Picard Tools -- samtools -- bwa -- pairix -- juicer +- [Picard Tools](https://broadinstitute.github.io/picard/) +- [samtools](https://www.htslib.org/) +- [bowtie2](https://bowtie-bio.sourceforge.net/bowtie2/index.shtml) +- [HiC-Pro](https://github.com/nservant/HiC-Pro) +- [juicer](https://github.com/aidenlab/juicer) +- [bedtools](https://github.com/arq5x/bedtools2) ## Reference Files @@ -56,12 +55,35 @@ The following reference files are used as the basis of the Hi-C Workflow: # > GRCh38_no_alt.fa: OK ``` -- The following command is used to prepare the BWA index file: +- The following command is used to prepare the bowtie2 index files: ```bash - bwa index GRCh38_no_alt.fa + bowtie2-build --threads=~{ncpu} ~{base} ~{prefix} ``` +- The Hi-C pipeline also uses a standard list of excluded regions. This can be +obtained using the following commands: + +```bash +wget https://github.com/Boyle-Lab/Blacklist/raw/master/lists/hg38-blacklist.v2.bed.gz +``` + +- The chromosome lengths are extracted from the reference using the following commands: + +```bash +samtools faidx -o GRCh38_no_alt.fa.fai GRCh38_no_alt.fa +cut -f 1,2 GRCh38_no_alt.fa.fai > chromsizes.tab +``` + +- A fragment file for each desired restriction site can also be extracted using HiC-Pro: + +```bash +digest_genome.py \ + -r ~{restriction_site} \ + -o ~{output_name} \ + ~{reference} +``` + ## Workflow Here are the resulting steps in the Hi-C workflow. There might be slight alterations in the actual implementation, which can be found in [the St. Jude Cloud workflows repository](https://github.com/stjudecloud/workflows/blob/master/workflows/hic/hic-standard.wdl). @@ -75,59 +97,587 @@ Here are the resulting steps in the Hi-C workflow. There might be slight alterat IGNORE=MISSING_PLATFORM_VALUE ``` -2. Run samtools `collate` on the validated BAM file. +2. Run samtools `quickcheck` on the BAM file. ```bash - samtools collate \ - -o $COLLATED_BAM \ + samtools quickcheck \ $INPUT_BAM ``` -3. Run Samtools `fastq` on the collated BAM file. +3. Run samtools `split` on the BAM file. ```bash + samtools split \ + --threads "$n_cores" \ + -u ~{prefix}.unaccounted_reads.bam \ + -f '~{prefix}.%!.bam' \ + ~{bam} + + ``` +4. Run samtools `collate` and `fastq` on split BAM files to create FASTQ files. + + ```bash + samtools collate \ + ~{bam} | samtools fastq \ - -1 $READ_ONE_GZ \ # Fastq with read 1s - -2 $READ_TWO_GZ \ # Fastq with read 2s - $COLLATED_BAM # Input BMA to convert to fastq + -1 $READ_ONE_GZ \ # Fastq with read 1s + -2 $READ_TWO_GZ \ # Fastq with read 2s + ``` + +5. Run fq `lint` to validate FASTQ files. + + ```bash + fq lint \ + ~{read_one_fastq} \ + ~{read_two_fastq} + ``` + +6. Extract read groups from the input BAM file. + + ```bash + BAM="~{bam}" OUTFILE="read_groups.json" python - <> cmds + done + ``` + +8. For each pair of read files, align with `bowtie2`. + + ```bash + bowtie2 \ + --trim5 0 \ + --trim3 0 \ + -N 0 \ + -L 30 \ + --dpad 15 \ + --gbar 4 \ + --end-to-end \ + --mp 6 \ + --np 1 \ + --rdg 5,3 \ + --rfg 5,3 \ + --minins 0 \ + --maxins 500 \ + --un-gz ~{unpaired_unaligned_reads} \ + --met-file ~{metrics_files} \ + --met 1 \ + --rg-id ~{read_group_id} \ + --rg ~{read_group_fields} \ # --rg is specified once per read group field, other than ID. + -p 1 \ + --reorder \ + --seed 0 \ + --score-min L,-0.600000,-0.200000 \ + -i S,1.000000,0.500000 \ + -D 20 \ + -R 3 \ + -x ~{BOWTIE2_DB} \ + -U ~{single_end_Fastq} \ + | samtools view -bS - > ~{global_bam} + ``` +9. If a ligation site was provided, filter the global alignment BAM to remove unmapped reads. + + ```bash + samtools view \ + --threads "$n_cores" \ + -hb \ + -F 0x4 \ + ~{global_alignment_bam} \ + > ~{global_mapped_only_bam} + ``` + +10. If a ligation site was provided, run HiC-Pro cutsite trimming. + + ```bash + cutsite_trimming \ + --fastq $fastq \ + --cutsite ~{ligation_site} \ + --out ~{trimmed_fastq} \ + > ~{trim_log} + + gzip ~{trimmed_fastq} && rm $fastq + ``` + +11. If a ligation site was provided, realign unmapped reads after trimming with `bowtie2`. + + ```bash + bowtie2 \ + --trim5 0 \ + --trim3 0 \ + -N 0 \ + -L 20 \ + --dpad 15 \ + --gbar 4 \ + --end-to-end \ + --mp 6 \ + --np 1 \ + --rdg 5,3 \ + --rfg 5,3 \ + --minins 0 \ + --maxins 500 \ + --met-file ~{metrics_files} \ + --met 1 \ + --rg-id ~{read_group_id} \ + --rg ~{read_group_fields} \ # --rg is specified once per read group field, other than ID. + -p 1 \ + --reorder \ + --seed 0 \ + --score-min L,-0.600000,-0.200000 \ + -i S,1.000000,0.500000 \ + -D 20 \ + -R 3 \ + -x ~{BOWTIE2_DB} \ + -U ~{unaligned_trimmed_fastq} \ + | samtools view -bS - > ~{local_aligned_bam} + ``` + +12. If a ligation site was provided, merge the realigned reads and the globally mapped reads. + + ```bash + samtools merge \ + --threads "$n_cores" \ + -n \ + -c \ + -p \ + ~{merged_bam} \ + ~{global_bam} ~{local_aligned_bam} + ``` + +13. Run samtools `sort` to sort the resulting BAM file. + + ```bash + samtools sort \ + --threads $n_cores \ + -l 6 \ + -m 2560M \ + -n \ + -o ~{sorted_bam} \ + ~{merged_bam} + ``` + +14. Compute mapping statistics for each read. Note: this is a reimplementation of https://github.com/nservant/HiC-Pro/blob/master/scripts/mapping_stat.sh + + ```bash + echo "## HiC-Pro Mapping Statistics" > ~{mapstat} + echo "## ~{mapstat}" >> ~{mapstat} + + total_reads=$(samtools view -c ~{sorted_bam}) + + mapped_reads=$(samtools view -c -F 4 ~{sorted_bam}) + + global_mapped_reads=$(samtools view -c -F 4 ~{global_bam}) + + if ~{local_bam} + then + local_mapped_reads=$(samtools view -c -F 4 ~{local_bam}) + fi + + echo -e "total_R1\t$total_reads" >> ~{mapstat} + echo -e "mapped_R1\t$mapped_reads" >> ~{mapstat} + echo -e "global_R1\t$global_mapped_reads" >> ~{mapstat} + echo -e "local_R1\t$local_mapped_reads" >> ~{mapstat} + ``` + +15. Merge the read ends with HiC-Pro's `mergeSAM.py`. + + ```bash + python mergeSAM.py \ + -q 10 \ + -t \ + -v \ + -f ~{read_one_sorted_bam} \ + -r ~{read_two_sorted_bam} \ + -o ~{paired_bam} + ``` + +16. Extract mapped Hi-C fragments with HiC-Pro's `mapped_2hic_fragments.py`. The fragment file is extracted from the genome FASTA and corresponds to the restriction site enzyme used in the Hi-C experiment. + + ```bash + python mapped_2hic_fragments.py -f ~{fragment_file} \ + -v \ + -r ~{paired_bam} \ + -S \ + -a + + LANG=en; sort -k2,2V -k3,3n -k5,5V -k6,6n -o ~{sorted_pairs} ~{pairs} + ``` + +17. Merge valid Hi-C interactions. Note: this is a reimplementation of https://github.com/nservant/HiC-Pro/blob/master/scripts/merge_valid_interactions.sh. + + ```bash + LANG=en; sort \ + -k2,2V -k3,3n -k5,5V -k6,6n \ + -m ~{valid_pairs_files} \ + | awk -F "\t" \ + 'BEGIN{c1=0;c2=0;s1=0;s2=0}(c1!=$2 || c2!=$5 || s1!=$3 || s2!=$6){print;c1=$2;c2=$5;s1=$3;s2=$6}' \ + > ~{all_valid_pairs} + + + allcount=$(cat ~{valid_pairs_files} | wc -l) + allcount_rmdup=$(cat ~{all_valid_pairs} | wc -l) + ndbup=$(( $allcount - $allcount_rmdup )) + + ## merge stat file + echo -e "valid_interaction\t"$allcount > ~{all_valid_pairs}.mergestat + echo -e "valid_interaction_rmdup\t"$allcount_rmdup >> ~{all_valid_pairs}.mergestat + awk 'BEGIN{cis=0;trans=0;sr=0;lr=0} $2 == $5{cis=cis+1; d=$6>$3?$6-$3:$3-$6; if (d<=20000){sr=sr+1}else{lr=lr+1}} $2!=$5{trans=trans+1}END{print "trans_interaction\t"trans"\ncis_interaction\t"cis"\ncis_shortRange\t"sr"\ncis_longRange\t"lr}' ~{all_valid_pairs} >> ~{all_valid_pairs}.mergestat + ``` +18. Merge stats. Note: this is a reimplementation of https://github.com/nservant/HiC-Pro/blob/master/scripts/merge_stats.sh. + + ```bash + mkdir read1 + ln -s ~{read_one_mapping_stats} read1/ + python /HiC-Pro_3.0.0/scripts/merge_statfiles.py \ + -d read1 \ + -p "*.R1*.mapstat" \ + -v \ + > ~{read_one}.mmapstat + + # merge read2 mapping stats + mkdir read2 + ln -s ~{read_two_mapping_stats} read2/ + python /HiC-Pro_3.0.0/scripts/merge_statfiles.py \ + -d read2 \ + -p "*.R2*.mapstat" \ + -v \ + > ~{read_two}.mmapstat + + # merge pairing stats + mkdir pairs + ln -s ~{pairing_stats} pairs/ + python /HiC-Pro_3.0.0/scripts/merge_statfiles.py \ + -d pairs \ + -p "*.pairstat" \ + -v \ + > ~{prefix}.mpairstat + + # merge RS stat + mkdir rsstat + ln -s ~{rs_stat_files} rsstat/ + python /HiC-Pro_3.0.0/scripts/merge_statfiles.py \ + -d rsstat \ + -p "*.RSstat" \ + -v \ + > ~{prefix}.mRSstat + ``` + +19. QC Hi-C results. Note: this is a reimplementation of https://github.com/nservant/HiC-Pro/blob/master/scripts/make_plots.sh. + + ```bash + mkdir plots + + mkdir bowtie + ln -sf ~{mapstat_files} bowtie/ + ln -sf ~{pairstat_files} bowtie/ + + mkdir hic + ln -sf ~{rs_stat_files} ~{valid_pair_files} hic/ + + mkdir stats + ln -sf ~{all_valid_pairs}.mergestat stats/ + + # mapping plots + if [[ "all" == "all" || "all" == "mapping" ]] + then + R CMD BATCH \ + --no-save \ + --no-restore \ + "--args picDir='plots' bwtDir='bowtie' sampleName='~{prefix}' r1tag='.R1' r2tag='.R2'" \ + /HiC-Pro_3.0.0/scripts/plot_mapping_portion.R \ + plot_mapping_portion.Rout + fi + + # pairing plots + if [[ "all" == "all" || "all" == "pairing" ]] + then + R CMD BATCH \ + --no-save \ + --no-restore \ + "--args picDir='plots' bwtDir='bowtie' sampleName='~{prefix}' rmMulti='1' rmSingle='1'" \ + /HiC-Pro_3.0.0/scripts/plot_pairing_portion.R \ + plot_pairing_portion.Rout + fi + + # filtering plots + if [[ "all" == "all" || "all" == "filtering" ]] + then + R CMD BATCH \ + --no-save \ + --no-restore \ + "--args picDir='plots' hicDir='hic' sampleName='~{prefix}' rmMulti='1' rmSingle='1'" \ + /HiC-Pro_3.0.0/scripts/plot_hic_fragment.R \ + plot_hic_fragment.Rout + fi + + # contacts plots + if [[ "all" == "all" || "all" == "contacts" ]] + then + R CMD BATCH \ + --no-save \ + --no-restore \ + "--args picDir='plots' hicDir='hic' statsDir='stats' sampleName='~{prefix}'" \ + /HiC-Pro_3.0.0/scripts/plot_hic_contacts.R \ + plot_hic_contacts.Rout + fi ``` -4. Run picard `FastqToSam` on fastq files to create unaligned BAM file. + +20. Build raw contact maps. + + ```bash + for bin_size in ~{sep(" ", bin_sizes)} + do + cat ~{hic_file} \ + | /HiC-Pro_3.0.0/scripts/build_matrix \ + --matrix-format ~{matrix_format} \ + ~{( + if (length(bin_sizes) > 1) + then "--binsize $bin_size" + else "--binfile ~{select_first([genome_fragment, ""])}" + )} \ + --chrsizes ~{chromsizes} \ + --ifile /dev/stdin \ + --oprefix ~{prefix}_${bin_size} + + done + ``` + +21. Run ICE normalization. + + ```bash + for map in ~{sep(" ", contact_counts)} + do + name=$(basename $map) + for bin in ~{sep(" ", bin_sizes)} + do + ice \ + --results_filename ${name}_${bin}_iced.matrix \ + --filter_low_counts_perc ~{filter_low_counts_percentage} \ + --filter_high_counts_perc ~{filter_high_counts_percentage} \ + --max_iter ~{max_iterations} \ + --eps ~{precision} \ + --remove-all-zeros-loci \ + --output-bias 1 \ + $map + done + done + ``` + +22. Convert Hi-C contacts to ".hic" file using HiC-Pro's `hicpro2juicebox.sh`. + + ```bash + /HiC-Pro_3.0.0/bin/utils/hicpro2juicebox.sh \ + -i ~{all_valid_pairs} \ + -g ~{chromsizes} \ + -j ~{juicer_tools_jar} + ``` + +23. If an exclude list was specified, filter results. + + ```bash + #left bed + awk -F "\t" -v OFS="\t" '{print $2,$3,$3,$1}' ~{all_valid_pairs} \ + | slopBed -i stdin -b ~{padding} -g ~{chromsizes} \ + | intersectBed -a stdin -b ~{base} -u > left.bed + + #right bed + awk -F "\t" -v OFS="\t" '{print $5,$6,$6,$1}' ~{all_valid_pairs} \ + | slopBed -i stdin -b ~{padding} -g ~{chromsizes} \ + | intersectBed -a stdin -b ~{base} -u > right.bed + + cat <(cut -f 4 left.bed) <(cut -f 4 right.bed)|sort -u > filter.pair + + python < ~{prefix}.allValidPairs.stats + ``` + +24. Write HiLOW-style QC report. + + ```bash + python < 80: + VERDICT[aligned] = "GOOD" + else: + VERDICT[aligned] = "BAD" + + if PERCENTAGES["valid_interactionPairs"] > 50: + VERDICT["valid_interactionPairs"] = "GOOD" + else: + VERDICT["valid_interactionPairs"] = "BAD" + + if PERCENTAGES["cis_shortRange"] > 50: + VERDICT["cis_shortRange"] = "BAD" + elif PERCENTAGES["cis_shortRange"] > 30: + VERDICT["cis_shortRange"] = "MARGINAL" + else: + VERDICT["cis_shortRange"] = "GOOD" + + if PERCENTAGES["cis_longRange"] > 40: + VERDICT["cis_longRange"] = "GOOD" + elif PERCENTAGES["cis_longRange"] > 20: + VERDICT["cis_longRange"] = "MARGINAL" + else: + VERDICT["cis_longRange"] = "BAD" + + REPORT = open("~{prefix}_QCreport.txt", 'w') + REPORT.write("STAT\tCOUNTS\tPERCENTAGE\tVERDICT\n") + REPORT.write("Total_pairs_processed\t" + str(RESULTS["Total_pairs_processed"]) + "\n") + for var in REP_VAR: + REPORT.write(var + "\t" + str(COUNTS[var]) + "\t" + str(PERCENTAGES[var]) + "\t" + VERDICT[var] + '\n') + + if os.path.isfile("~{peaks_bed}"): + REPORT.write("peaks\t" + str(PEAKS) + "\n") + if os.path.isfile('~{fithichip_bed}'): + REPORT.write("loops\t" + str(LOOPS) + "\n") + REPORT.write("loops_significant\t" + str(LOOPS_SIGNIFICANT) + "\n") + CODE + ``` + +25. Merge per read group BAMs with samtools `merge`. + + ```bash + samtools merge \ + --threads "$n_cores" \ + -c \ + -p \ + ~{combined_bam} \ + ~{per_read_group_bams} + ``` + +26. Run samtools `fixmate` to update mate information. ```bash - picard FastqToSam \ - FASTQ=$READ_ONE_GZ \ # Fastq with read 1s - FASTQ2=$READ_TWO_GZ \ # Fastq with read 2s - OUTPUT=$UNALIGNED_BAM # Output unaligned BAM file + samtools fixmate \ + --threads "$n_cores" \ + -c \ + -m \ + ~{combined_bam} \ + ~{fixmate_bam} ``` -5. Run bwa `mem` on fastq files to create aligned BAM file. +27. Run samtools `sort` to coordinate-sort the BAM. ```bash - bwa mem \ - $BWA_DB \ # BWA reference database - $READ_ONE_GZ \ # Fastq with read 1s - $READ_TWO_GZ \ # Fastq with read 2s - | samtools view -hb \ # Convert output SAM to BAM - > $ALIGNED_BAM + samtools sort \ + --threads $n_cores \ + -l 6 \ + -m 3584M \ + -o ~{sorted_bam} \ + ~{fixmate_bam} ``` -6. Run pairix `bam2pairs` on aligned BAM file. +28. Run samtools `markdup`. ```bash - bam2pairs \ - $ALIGNED_BAM \ # BAM file from bwa mem - $PAIRS # Output mapped read pairs + samtools markdup \ + --threads "$n_cores" \ + -f "~{prefix}.txt" \ + --read-coords '[!-9;-?A-~:]+:([!-9;-?A-~]+):([0-9]+):([0-9]+)' \ + --coords-order txy \ + -S \ + --no-multi-dup \ + -l 300 \ + -d 0 \ + -c \ + ~{sorted_bam} \ + ~{prefix}.bam ``` -7. Run Juicer `pre` to generate .hic file. +29. Index the final BAM with samtools `index`. ```bash - juicer_tools pre \ - -f $RESTRICTION_SITES \ # List of restriction sites, if available - $PAIRS \ # Reads with both mates mapped - $HIC \ # hic file with multiple resolutions - $GENOME_ID # Predefined genome matching alignment + samtools index --threads "$n_cores" ~{prefix}.bam ~{prefix}.bam.bai ``` # Further Reading From b3178902558ada2f9e4035ee7f89d77321c192f6 Mon Sep 17 00:00:00 2001 From: Andrew Thrasher Date: Fri, 10 Jan 2025 15:29:25 -0500 Subject: [PATCH 10/10] chore: outstanding language change --- .vscode/settings.json | 9 ++++++--- text/hic-workflow.md | 2 +- 2 files changed, 7 insertions(+), 4 deletions(-) diff --git a/.vscode/settings.json b/.vscode/settings.json index b2e8b62..75c814c 100644 --- a/.vscode/settings.json +++ b/.vscode/settings.json @@ -3,10 +3,13 @@ "editor.formatOnSave": true, "editor.wordWrap": "wordWrapColumn", "editor.wordWrapColumn": 80, - "editor.wordBasedSuggestions": false, + "editor.wordBasedSuggestions": "off", "markdown.extension.toc.levels": "1..1", "[markdown]": { "editor.wordWrap": "wordWrapColumn", "editor.wordWrapColumn": 80 - } -} + }, + "cSpell.ignoreWords": [ + "GIAB" + ] +} \ No newline at end of file diff --git a/text/hic-workflow.md b/text/hic-workflow.md index 4baf509..3cb843d 100644 --- a/text/hic-workflow.md +++ b/text/hic-workflow.md @@ -86,7 +86,7 @@ digest_genome.py \ ## Workflow -Here are the resulting steps in the Hi-C workflow. There might be slight alterations in the actual implementation, which can be found in [the St. Jude Cloud workflows repository](https://github.com/stjudecloud/workflows/blob/master/workflows/hic/hic-standard.wdl). +Here are the resulting steps in the Hi-C workflow. There might be slight alterations in the actual implementation, which can be found in [the St. Jude Cloud workflows repository](https://github.com/stjudecloud/workflows/blob/master/workflows/hic/hic-standard.wdl). The workflow primarily consists of a WDL reimplementation of [HiC-Pro](https://github.com/nservant/HiC-Pro/tree/master). 1. Run `picard ValidateSam` on the incoming BAM to ensure that it is well-formed enough to strip alignment information.

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