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feat: added per-read anchor requirement to junction extract #195

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TimD1
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@TimD1 TimD1 commented Sep 19, 2024

@TimD1
fix: CMake build errors on Mac resolved
435e8b8 
 - closes griffithlab#176, CMake was including `src/utils/bedtools/gzstream/version`
   as a C++ source file, when it was a simple text file containing version info
 - I moved this version info into `src/utils/bedtools/gzstream/README`
@TimD1
fix: min_intron_length_ initialization in JunctionsExtractor
1260bf9 
 - fixes griffithlab#188, min_intron_length_ was accidentally set from min_anchor_len_
@TimD1
feat: added per-read flag and map quality filtering
409a492 
 - closes griffithlab#183, flag based filtering
 - closes griffithlab#189, mapping quality based filtering
 - option '-F' filters reads containing any of these flags
 - option '-f' filters reads not containing all these flags
 - option '-q' filters reads below this mapping quality
@TimD1
feat: added per-read anchor requirement to junction extract
1e3080c 
 - closes griffithlab#186, reads now only 'support' a junction if they have at least
   a given minimum anchor length, supplied with the '-A' flag (default 0)
@santataRU
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Hi, All,
The per-read anchor filtering via the -A flag works as expected, but the -a option doesn’t appear to work in regtools-187 (v1.1.0). Is the -a option obsolete?

I tested with a small size bam (link: https://drive.google.com/drive/folders/173RVTDZor_zWy-3SRz_RYDcg-2m9YjZx?usp=sharing) and results below:

Program: regtools
Version: 1.1.0
Usage: regtools junctions extract [options] indexed_alignments.bam
Options:
-a INT Minimum anchor length. Junctions which satisfy a minimum
anchor length on both sides are reported. [8]
-A INT Minimum read anchor length. Reads which satisfy a minimum
anchor length on both sides 'support' a junction. [0]
-m INT Minimum intron length. [70]
-M INT Maximum intron length. [500000]
-f INT Only use alignments where all flag bits set here are set. [0]
-F INT Only use alignments where no flag bits set here are set. [0]
-q INT Only use alignments with this mapping quality or above. [0]
-o FILE The file to write output to. [STDOUT]
-r STR The region to identify junctions
in "chr:start-end" format. Entire BAM by default.
-s INT Strandness mode
XS, use XS tags provided by aligner; RF, first-strand; FR, second-strand. REQUIRED
-t STR Tag used in bam to label strand. [XS]
-b STR The file containing the barcodes of interest for single cell data.

Please supply strandness mode with '-s' option!

(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/HT1080_shNT-2_VSVNL43_sorted_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 0
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/HT1080_shNT-2_VSVNL43_sorted_junction_743_5917.bam
Output file: NA

NL43_Gallardo 621 6012 JUNC00000001 2 + 621 6012 255,0,0 2 122,96 0,5295
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 0
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 605 6065 JUNC00000001 9 + 605 6065 255,0,0 2 138,149 0,5311
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -a 8 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 0
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 605 6065 JUNC00000001 9 + 605 6065 255,0,0 2 138,149 0,5311
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -a 15 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 15
Minimum read anchor length: 0
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 605 6065 JUNC00000001 9 + 605 6065 255,0,0 2 138,149 0,5311
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -a 100 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 100
Minimum read anchor length: 0
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 605 6065 JUNC00000001 9 + 605 6065 255,0,0 2 138,149 0,5311
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -A 100 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 100
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -A 10 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 10
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 621 6012 JUNC00000001 2 + 621 6012 255,0,0 2 122,96 0,5295
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -A 8 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 8
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 621 6012 JUNC00000001 2 + 621 6012 255,0,0 2 122,96 0,5295
(base) bieniaszlab@pauls-imac-2 build % ./regtools junctions extract -A 2 -s RF /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam

Program: regtools
Version: 1.1.0
Minimum junction anchor length: 8
Minimum read anchor length: 2
Minimum intron length: 70
Maximum intron length: 500000
Require alignment flags: 0
Filter alignment flags: 0
Minimum alignment mapping quality: 0
Alignment: /Volumes/Xiao_lab3/RNAseq/20250410_HT1080_shDDX42_shDDX46_two_shRNAs_VSVNL43_5dpt/aviti/trim_fastq/cutadapt_trim_fastq/NL43_garllardo_map/mapped_bam/unique_mapped/HT1080_shNT-2_VSVNL43_mapped_unique_mapped_junction_743_5917.bam
Output file: NA

NL43_Gallardo 605 6064 JUNC00000001 6 + 605 6064 255,0,0 2 138,148 0,5311

@santataRU santataRU mentioned this pull request May 6, 2025
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Feature request: add per-read anchor requirement to junction extract tool
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@TimD1 @santataRU