Version Compatibility

Reference examples tested with: bedtools 2.31+, pybedtools 0.10+, pyranges 0.x (the pyranges1 rewrite ships as a separate package), samtools 1.19+, pandas 2.2+, UCSC liftOver / CrossMap 0.7+ (the bare CrossMap entry point replaced CrossMap.py at 0.7.0).

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags
• Python: pip show <package> then help(module.function) to check signatures

pyranges has a major-version API split: pyranges 0.x and the pyranges1 rewrite differ in method names and DataFrame access; both keep Chromosome/Start/End columns and 0-based half-open coordinates. Check import pyranges; pyranges.__version__ before chaining methods. Operations that need chromosome lengths (slop, complement, shuffle, makewindows -g, -sorted ordering) require a genome/chrom.sizes file. If code throws an error, introspect the installed tool and adapt rather than retrying.

BED File Basics

"Work with this interval file without shifting everything by one base" -> Establish the coordinate convention from the format, read/create/validate/sort the intervals, and convert across format boundaries with the correct base shift.

• CLI: bedtools sort -i in.bed, bedtools getfasta, bedtools makewindows -g genome.txt -w 10000, sort -k1,1 -k2,2n
• Python: pybedtools.BedTool('in.bed'), pr.read_bed('in.bed') (pyranges), pd.read_csv(sep='\t', comment='#')

The Single Most Important Modern Insight -- A Coordinate Is a Bare Integer With No Self-Describing Convention

A start column is just an int. Nothing in the file says whether it is 0-based or 1-based, so the convention lives in the analyst's head, keyed off the format, not the data. BED is 0-based half-open [start, end); GTF/GFF, SAM, VCF, and wiggle are 1-based fully-closed [start, end]. Three load-bearing consequences:

1. The conversion is start - 1, end unchanged -- and it throws no error if wrong. A botched convention shift still parses, still runs, and silently shifts every answer by one base: gene bodies 1 bp short, boundary SNPs flipping in/out, exact-edge intersections toggling. The end is numerically identical between BED-half-open and GFF-closed because GFF's last included base and BED's first excluded position are the same boundary. The symmetry instinct (subtract 1 from both) is the classic bug. The reflex: convert start_bed = start_1based - 1 (end unchanged) and test the round-trip on a 1 bp feature -- BED chr1 5 6 == GFF chr1 6 6, both one base. Length is end - start in BED (no +1); end - start + 1 in GTF.

2. The two truly silent file failures. (a) Chrom-name mismatch (chr1 vs 1, chrM vs MT): intersecting a chr-prefixed file against a bare-numeral one yields a perfectly valid empty result -- "no overlap" looks like biology, not a bug. Confirm shared naming (cut -f1 a.bed | sort -u) before any cross-file op. (b) CRLF line endings from Excel/Windows glue \r onto the last field (end becomes "100\r"); the tell is "works for some tools, breaks for others." cat -A shows ^M$; fix with dos2unix. Never open a BED in Excel -- it date-mangles SEPT9->9-Sep and float-truncates large coordinates.

3. bedtools -sorted assumes both inputs share the SAME chromosome order. With a lexicographic (chr1, chr10, chr2) vs version (chr1, chr2, chr10) sort mismatch, modern bedtools (>=~2.25) detects the inconsistency and errors out (exit 1, chromomsome sort ordering ... is inconsistent); older versions silently swept past and dropped chr10-chr22. Either sort both files with the identical command, or pass -g genome.txt (derived from the same reference FASTA) to pin the expected order. For one-off work, omit -sorted (the in-memory path tolerates any order). The mismatch that stays SILENT on every version is a chromosome-NAME difference (chr1 vs 1), which returns an empty result with no error.

Tool Taxonomy

Tool	Role	Mechanism	When
bedtools	CLI interval algebra (Quinlan 2010 Bioinformatics 26:841)	streaming, sorted-input reference implementation	shell pipelines, large files, reproducible one-liners
pybedtools	Python wrapper over bedtools (Dale 2011 Bioinformatics 27:3423)	shells out per op; BedTool objects + iterators	inside a Python analysis; chaining with pandas
pyranges	pure-Python interval engine (Stovner 2020 Bioinformatics 36:918)	vectorized PyRanges/pandas, no bedtools binary	large in-memory joins, dataframe-native workflows
pandas	flat tabular read	read_csv(sep='\t'); knows NO coordinate semantics	quick filter/inspect; the analyst enforces 0-based + sort manually
UCSC bedToBigBed / tabix	indexed/compressed BED for random access	requires sort -k1,1 -k2,2n, no track lines	browser tracks, region queries on huge files

Decision Tree by Scenario

Scenario	Recommended	Why
Quick create/sort/filter on the command line	bedtools + coreutils sort -k1,1 -k2,2n	no Python overhead; reproducible
Inside a pandas/Python pipeline	pybedtools or pyranges	stays in-process; pyranges if no bedtools binary
Convert VCF/GTF/SAM positions to BED	subtract 1 from start, end unchanged	the convention boundary; test on a 1 bp feature
Empty intersect / "no overlap found"	check chrom naming (chr1 vs 1) FIRST	the most common silent null result
Using -sorted for speed/RAM	sort both files identically, or pass -g genome.txt	modern bedtools errors on a lexicographic-vs-version mismatch; old versions dropped chroms silently
Need chromosome lengths (slop/complement/windows)	generate genome.txt from the SAME FASTA	a stale/generic chrom.sizes rots slop/complement
Set operations on these intervals	-> interval-arithmetic	this skill is the format/coordinate substrate
Parse a GTF/GFF gene model	-> gtf-gff-handling	1-based, parent/child hierarchy, not a flat BED
Peaks not yet called	-> chip-seq/peak-calling or atac-seq/atac-peak-calling	this category operates on existing intervals
Convert between assemblies (hg19<->hg38)	liftOver/CrossMap, report unmapped	a different problem from convention shifts

BED Columns (BED3 -> BED12)

The first 3 fields are required; the rest are optional but positional (cannot supply field N without 1..N-1), and the field count must be identical on every line.

BED3   chrom  start  end
BED4   + name
BED5   + score (int 0-1000; '.' allowed)
BED6   + strand (+/-/.)            # the common stranded-interval form
BED12  + thickStart thickEnd itemRgb blockCount blockSizes blockStarts   # transcript/exon models

narrowPeak is BED6+4 (signalValue pValue qValue peak); broadPeak is BED6+3 (drops peak). The peak column is a 0-based offset from chromStart (absolute summit = chromStart + peak), -1 if none; pValue/qValue are -log10 scaled with -1 meaning "not assigned", NOT p=0.1.

Create and Read BED Files

import pybedtools
import pandas as pd

intervals = [('chr1', 100, 200, 'peak1', 100, '+'), ('chr1', 300, 400, 'peak2', 200, '-')]
bed = pybedtools.BedTool(intervals)                                       # from list of tuples
bed = pybedtools.BedTool.from_dataframe(pd.read_csv('peaks.tsv', sep='\t'))  # from a DataFrame
bed.saveas('peaks.bed')

for iv in pybedtools.BedTool('peaks.bed'):
    print(iv.chrom, iv.start, iv.end, len(iv))   # start/end are ints; len(iv) == end - start
df = pybedtools.BedTool('peaks.bed').to_dataframe(names=['chrom', 'start', 'end', 'name', 'score', 'strand'])

pandas reads BED as a flat table but knows nothing about coordinates: pd.read_csv('in.bed', sep='\t', header=None, comment='#') -- the analyst enforces 0-based and sorts manually.

Generate the Genome / chrom.sizes File

Goal: Produce the authoritative chromosome-length file that slop, complement, shuffle, makewindows -g, and -sorted ordering depend on.

Approach: Index the exact reference FASTA the rest of the pipeline used and take its first two columns -- never download a generic hg38.chrom.sizes and hope it matches the assembly the BAM was aligned to.

samtools faidx ref.fa
cut -f1,2 ref.fa.fai > genome.txt   # chrom<TAB>size; bedtools reads only the first 2 cols, accepts the .fai directly

Convert Across Format Boundaries

Goal: Move positions between BED and VCF/SAM/GTF/BAM without an off-by-one.

Approach: Subtract 1 from the 1-based start (end unchanged) going TO BED; for BAM, let bedtools do the conversion; verify a known single-base landmark afterwards.

grep -v '^#' in.vcf | awk 'BEGIN{OFS="\t"} {print $1, $2-1, $2}' > variants.bed   # VCF POS (1-based) -> BED; start-1
bedtools bamtobed -i in.bam > alignments.bed                                       # bedtools handles the convention
bedtools bamtobed -i in.bam -split > spliced.bed                                   # split spliced reads into blocks
bedtools getfasta -fi ref.fa -bed in.bed -name -fo out.fa                          # extract sequence (uses the .fai)

Sort, Validate, and Make Windows

bedtools sort -i in.bed > sorted.bed                 # lexicographic, == sort -k1,1 -k2,2n
bedtools sort -i in.bed -faidx names.txt > sorted.bed  # reorder to an arbitrary reference contig order
awk -F'\t' '{print NF}' in.bed | sort -u             # field count consistent? (one value expected)
awk -F'\t' '$2 < 0 || $2 >= $3' in.bed               # negative or inverted intervals (bedtools also errors on these)
cut -f1 in.bed | sort -u                             # chromosome names (compare against the partner file)
bedtools makewindows -g genome.txt -w 10000 -s 5000 -i winnum > windows.bed   # 10 kb sliding windows, step 5 kb, numbered

Cross-Assembly Liftover (a different problem from convention conversion)

Goal: Move coordinates between genome builds (hg19<->hg38, mm10<->mm39) - which is remapping to a different reference, NOT the 0-based/1-based convention shift above.

Approach: Map intervals through a chain file with UCSC liftOver (BED) or CrossMap (BED/VCF/GFF/BAM/bigWig), and ALWAYS inspect the unmapped file - regions that fail to map (assembly gaps, rearrangements, split/merged contigs) are dropped, and silently ignoring them biases everything downstream.

liftOver in.hg19.bed hg19ToHg38.over.chain.gz out.hg38.bed unmapped.bed   # UCSC; chain from UCSC goldenPath
wc -l unmapped.bed                                                         # NEVER skip: dropped regions are not random
CrossMap bed GRCh37_to_GRCh38.chain.gz in.bed > out.bed                    # CrossMap also does vcf/gff/bam/bigwig

A coordinate is meaningless without its assembly just as it is meaningless without its convention - record the build (and the chain provenance) alongside the file. Liftover is many-to-one and one-to-none in places; never assume a 1:1 round-trip.

BED12 Block Invariants

BED12 is a referentially-integral structure, not a flat table. blockStarts are offsets from chromStart, not absolute coordinates; the first blockStart must be 0; blockStarts[last] + blockSizes[last] must equal chromEnd - chromStart; blocks are ascending and non-overlapping. thickStart/thickEnd (the CDS) are absolute and independent of the blocks -- a non-coding feature sets both to chromStart. Validate by reconstructing absolute exon coordinates (chromStart + blockStarts[i]) and confirming they fall inside [chromStart, chromEnd]; bedtools bed12tobed6 explodes a model into per-exon BED6 as a quick sanity reconstruction.

Per-Method Failure Modes

Off-by-one at a convention boundary

Trigger: treating a GTF/VCF 1-based start as a BED chromStart. Mechanism: the convention is not stored in the data. Symptom: every feature 1 bp short; boundary intersections flip; no error. Fix: start - 1, end unchanged; test the round-trip on a 1 bp feature.

Chrom-name mismatch

Trigger: intersecting chr-prefixed against bare-numeral files. Mechanism: chrom strings never match. Symptom: valid empty/zero result presented as biology. Fix: harmonize naming across all files and genome.txt before any cross-file op.

Lexicographic-vs-version sort under -sorted

Trigger: two inputs sorted in different chromosome orders. Mechanism: sweep-line assumes one shared order and concludes chroms passed each other. Symptom: modern bedtools errors out (chromomsome sort ordering ... is inconsistent, exit 1); pre-2.25 silently dropped chr10-chr22 and favored low chroms. Fix: sort both identically or pass -g genome.txt; omit -sorted for one-off work.

CRLF line endings

Trigger: file authored/round-tripped through Windows/Excel. Mechanism: \r glues onto the last field. Symptom: "not an integer" or cryptic last-column errors; works in some tools. Fix: dos2unix or sed 's/\r$//'; never edit BED in Excel.

Stale / generic genome file

Trigger: a downloaded chrom.sizes that does not match the aligned FASTA. Mechanism: missing contigs or wrong lengths. Symptom: slop/complement run off real ends or drop contigs; -sorted order breaks. Fix: regenerate from the exact reference FASTA.

pyranges 0.x idioms on pyranges1

Trigger: running 0.x method/attribute names against the pyranges1 rewrite. Mechanism: the rewrite renamed methods and changed DataFrame access. Symptom: AttributeError. Fix: check pyranges.__version__ and use the matching API.

Quantitative Thresholds

Threshold	Source	Rationale
Required minimum 3 columns (BED3)	UCSC/hts-specs BEDv1	chrom/start/end; field count identical on every line
BED score integer 0-1000	UCSC spec	maps to browser gray shade; analysis tools tolerate out-of-range, browser clamps
narrowPeak/broadPeak -1 in pValue/qValue/peak	ENCODE narrowPeak.as	means "not assigned", NOT a real value (e.g. p=0.1 or summit at offset 0)
Convention shift: start - 1, end unchanged	BED 0-based vs GFF/VCF 1-based	only the start moves; ends coincide at the shared boundary
makewindows window/step (e.g. -w 10000 -s 5000)	analysis choice	resolution vs file size; state the size used, do not default silently
tabix BED needs -p bed (or -0)	tabix default is 1-based	-p bed sets chrom/start/end cols and 0-based interpretation

Common Errors

Error / symptom	Cause	Solution
Empty intersect / "no overlap"	chrom naming mismatch (chr1 vs 1, chrM vs MT)	harmonize naming across all files + genome.txt
Every feature 1 bp short	converted 1-based->BED without the start-1 (or subtracted from both)	start - 1, end unchanged; verify on a 1 bp feature
Every coordinate shifted one column right	UCSC MySQL/SELECT * table dump prepends a bin column (hierarchical binning index, Kent 2002)	drop field 1 before feeding bedtools: cut -f2-
-sorted errors or (old bedtools) favors low chroms	lexicographic vs version sort mismatch	sort both identically or pass -g genome.txt; or drop -sorted
"not an integer" on the last column	CRLF line endings	dos2unix / sed 's/\r$//'
Negative start / past-chrom-end after slop	missing or stale genome file	regenerate genome.txt from the aligned FASTA
bedToBigBed/tabix errors	unsorted input or track lines present	sort -k1,1 -k2,2n; strip track/browser/# lines
pyranges AttributeError	0.x vs pyranges1 API mismatch	check pyranges.__version__, use matching method names

References

• Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841-842.
• Dale RK, Pedersen BS, Quinlan AR. 2011. Pybedtools: a flexible Python library for manipulating genomic datasets and annotations. Bioinformatics 27:3423-3424.
• Stovner EB, Sætrom P. 2020. PyRanges: efficient comparison of genomic intervals in Python. Bioinformatics 36:918-919.
• Kent WJ, Sugnet CW, Furey TS, et al. 2002. The Human Genome Browser at UCSC. Genome Res 12:996-1006.
• The Browser Extensible Data (BED) format, hts-specs BEDv1. samtools.github.io/hts-specs/BEDv1.pdf.
• UCSC Genome Browser FAQ: Data File Formats. genome.ucsc.edu/FAQ/FAQformat.html.

Related Skills

• interval-arithmetic - Set operations (intersect/merge/subtract) on the intervals defined here
• gtf-gff-handling - 1-based annotation parsing and the parent/child gene-model hierarchy
• coverage-analysis - Per-base depth that becomes bedGraph intervals
• alignment-files/sam-bam-basics - BAM-to-BED conversion and the SAM-1-based vs BAM-0-based distinction
• variant-calling/vcf-basics - VCF POS (1-based) to BED conversion and indel left-anchoring