pileup-generation

Name: pileup-generation
Author: GPTomics

1.1kstars185forks

How to install pileup-generation?

gptomics/bioskills/pileup-generation

$npx -y skills add GPTomics/bioSkills --skill pileup-generation

Installs into the current project.

Summary

Generate pileup data for variant calling using samtools mpileup and pysam. Use when preparing data for variant calling, analyzing per-position read data, or calculating allele frequencies.

Files · 1

View on GitHub

SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: bcftools 1.19+, pysam 0.22+, samtools 1.19+
4
5	Before using code patterns, verify installed versions match. If versions differ:
6	- Python: `pip show <package>` then `help(module.function)` to check signatures
7	- CLI: `<tool> --version` then `<tool> --help` to confirm flags
8
9	If code throws ImportError, AttributeError, or TypeError, introspect the installed
10	package and adapt the example to match the actual API rather than retrying.
11
12	# Pileup Generation
13
14	Generate pileup data for variant calling and position-level analysis.
15
16	"Generate pileup from BAM" -> Produce per-position read summaries showing depth, bases, and qualities.
17	- CLI: `samtools mpileup -f ref.fa input.bam`
18	- Python: `bam.pileup(chrom, start, end)` (pysam)
19
20	"Count alleles at a position" -> Extract per-base read support at a specific genomic coordinate.
21	- Python: iterate `pileup_column.pileups` and count bases (pysam)
22
23	## What is Pileup?
24
25	Pileup shows all reads covering each position in the reference, used for:
26	- Variant calling (with bcftools)
27	- Coverage analysis
28	- Allele frequency calculation
29	- SNP/indel detection
30
31	## samtools mpileup vs bcftools mpileup (Deprecation)
32
33	`samtools mpileup -g/-u` (BCF output for variant calling) was deprecated in samtools 1.9 and removed in 1.15 (the option no longer exists; the usage/manpage directs users to `bcftools mpileup`) -- the genotype-likelihood code now lives in `bcftools mpileup`, which keeps mpileup logic versioned alongside `bcftools call` and avoids version-skew bugs.
34
35	\| Use case \| Recommended tool \|
36	\|----------\|------------------\|
37	\| Quick allele counts at known sites \| `samtools mpileup` or pysam pileup \|
38	\| Germline variant calling (small genomes, simple cohorts) \| `bcftools mpileup` -> `bcftools call` \|
39	\| Germline WGS / WES production \| DeepVariant or HaplotypeCaller (not mpileup) \|
40	\| Somatic SNV/indel \| Mutect2 / VarDict / VarScan2 (direct from BAM) \|
41	\| Long-read small variants \| clair3 / DeepVariant ONT (direct from BAM) \|
42	\| Long-read SV \| Sniffles / cuteSV (direct from BAM) \|
43	\| Ultra-low-frequency (ctDNA / MRD) \| fgbio consensus -> `bcftools call` or hot-spot Mutect2 \|
44	\| Per-position allele counts (custom) \| pysam pileup \|
45
46	`samtools mpileup` (without `-g`) is still the standard tool for human-readable per-position read summaries.
47
48	### Basic Pileup
49	```bash
50	samtools mpileup -f reference.fa input.bam > pileup.txt
51	```
52
53	### Pileup Specific Region
54	```bash
55	samtools mpileup -f reference.fa -r chr1:1000000-2000000 input.bam
56	```
57
58	### Regions from BED
59	```bash
60	samtools mpileup -f reference.fa -l targets.bed input.bam
61	```
62
63	### Multiple BAM Files
64	```bash
65	samtools mpileup -f reference.fa sample1.bam sample2.bam sample3.bam > pileup.txt
66	```
67
68	## Output Format
69
70	Text pileup format (6 columns per sample):
71	```
72	chr1 1000 A 15 ............... FFFFFFFFFFF
73	chr1 1001 T 12 ............ FFFFFFFFFFFF
74	```
75
76	\| Column \| Description \|
77	\|--------\|-------------\|
78	\| 1 \| Chromosome \|
79	\| 2 \| Position (1-based) \|
80	\| 3 \| Reference base \|
81	\| 4 \| Read depth \|
82	\| 5 \| Read bases \|
83	\| 6 \| Base qualities \|
84
85	### Read Bases Encoding
86
87	\| Symbol \| Meaning \|
88	\|--------\|---------\|
89	\| `.` \| Match on forward strand \|
90	\| `,` \| Match on reverse strand \|
91	\| `ACGT` \| Mismatch (uppercase = forward) \|
92	\| `acgt` \| Mismatch (lowercase = reverse) \|
93	\| `^Q` \| Start of read (Q = MAPQ as ASCII) \|
94	\| `$` \| End of read \|
95	\| `+NNN` \| Insertion of N bases \|
96	\| `-NNN` \| Deletion of N bases \|
97	\| `*` \| Deleted base \|
98	\| `>` / `<` \| Reference skip (intron) \|
99
100	## Quality Filtering Options
101
102	### Minimum Mapping Quality
103	```bash
104	samtools mpileup -f reference.fa -q 20 input.bam
105	```
106
107	### Minimum Base Quality
108	```bash
109	samtools mpileup -f reference.fa -Q 20 input.bam
110	```
111
112	### Combined Quality Filters
113	```bash
114	samtools mpileup -f reference.fa -q 20 -Q 20 input.bam
115	```
116
117	### Maximum Depth (Critical Trap)
118	```bash
119	# samtools mpileup default -d 8000 silently truncates targeted / mt-DNA / amplicon / UMI-deduped data
120	# bcftools mpileup default -d 250 is far lower; both must be set explicitly when piping
121	samtools mpileup -f reference.fa -d 0 input.bam # no cap
122	samtools mpileup -f reference.fa -d 1000000 input.bam # explicit high cap
123
124	# WRONG -- samtools 8000 cap, then bcftools 250 cap re-applied
125	samtools mpileup -f ref.fa in.bam \| bcftools call -mv
126
127	# RIGHT -- single tool, explicit -d
128	bcftools mpileup -d 1000000 -f ref.fa in.bam \| bcftools call -mv
129	```
130
131	## BAQ: Base Alignment Quality (Critical Default)
132
133	When `-f ref.fa` is passed, BAQ is enabled by default. BAQ Phred-scales the probability that a base is misaligned (HMM realignment over a small window) and reduces base quality near indels. Tradeoffs: ~30% slower; suppresses FP SNVs

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pileup-generation

1.1kstars185forks

View on GitHub

How to install pileup-generation?

gptomics/bioskills/pileup-generation

$npx -y skills add GPTomics/bioSkills --skill pileup-generation

Installs into the current project.

Summary

Generate pileup data for variant calling using samtools mpileup and pysam. Use when preparing data for variant calling, analyzing per-position read data, or calculating allele frequencies.

Files · 1

View on GitHub

SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: bcftools 1.19+, pysam 0.22+, samtools 1.19+
4
5	Before using code patterns, verify installed versions match. If versions differ:
6	- Python: `pip show <package>` then `help(module.function)` to check signatures
7	- CLI: `<tool> --version` then `<tool> --help` to confirm flags
8
9	If code throws ImportError, AttributeError, or TypeError, introspect the installed
10	package and adapt the example to match the actual API rather than retrying.
11
12	# Pileup Generation
13
14	Generate pileup data for variant calling and position-level analysis.
15
16	"Generate pileup from BAM" -> Produce per-position read summaries showing depth, bases, and qualities.
17	- CLI: `samtools mpileup -f ref.fa input.bam`
18	- Python: `bam.pileup(chrom, start, end)` (pysam)
19
20	"Count alleles at a position" -> Extract per-base read support at a specific genomic coordinate.
21	- Python: iterate `pileup_column.pileups` and count bases (pysam)
22
23	## What is Pileup?
24
25	Pileup shows all reads covering each position in the reference, used for:
26	- Variant calling (with bcftools)
27	- Coverage analysis
28	- Allele frequency calculation
29	- SNP/indel detection
30
31	## samtools mpileup vs bcftools mpileup (Deprecation)
32
33	`samtools mpileup -g/-u` (BCF output for variant calling) was deprecated in samtools 1.9 and removed in 1.15 (the option no longer exists; the usage/manpage directs users to `bcftools mpileup`) -- the genotype-likelihood code now lives in `bcftools mpileup`, which keeps mpileup logic versioned alongside `bcftools call` and avoids version-skew bugs.
34
35	\| Use case \| Recommended tool \|
36	\|----------\|------------------\|
37	\| Quick allele counts at known sites \| `samtools mpileup` or pysam pileup \|
38	\| Germline variant calling (small genomes, simple cohorts) \| `bcftools mpileup` -> `bcftools call` \|
39	\| Germline WGS / WES production \| DeepVariant or HaplotypeCaller (not mpileup) \|
40	\| Somatic SNV/indel \| Mutect2 / VarDict / VarScan2 (direct from BAM) \|
41	\| Long-read small variants \| clair3 / DeepVariant ONT (direct from BAM) \|
42	\| Long-read SV \| Sniffles / cuteSV (direct from BAM) \|
43	\| Ultra-low-frequency (ctDNA / MRD) \| fgbio consensus -> `bcftools call` or hot-spot Mutect2 \|
44	\| Per-position allele counts (custom) \| pysam pileup \|
45
46	`samtools mpileup` (without `-g`) is still the standard tool for human-readable per-position read summaries.
47
48	### Basic Pileup
49	```bash
50	samtools mpileup -f reference.fa input.bam > pileup.txt
51	```
52
53	### Pileup Specific Region
54	```bash
55	samtools mpileup -f reference.fa -r chr1:1000000-2000000 input.bam
56	```
57
58	### Regions from BED
59	```bash
60	samtools mpileup -f reference.fa -l targets.bed input.bam
61	```
62
63	### Multiple BAM Files
64	```bash
65	samtools mpileup -f reference.fa sample1.bam sample2.bam sample3.bam > pileup.txt
66	```
67
68	## Output Format
69
70	Text pileup format (6 columns per sample):
71	```
72	chr1 1000 A 15 ............... FFFFFFFFFFF
73	chr1 1001 T 12 ............ FFFFFFFFFFFF
74	```
75
76	\| Column \| Description \|
77	\|--------\|-------------\|
78	\| 1 \| Chromosome \|
79	\| 2 \| Position (1-based) \|
80	\| 3 \| Reference base \|
81	\| 4 \| Read depth \|
82	\| 5 \| Read bases \|
83	\| 6 \| Base qualities \|
84
85	### Read Bases Encoding
86
87	\| Symbol \| Meaning \|
88	\|--------\|---------\|
89	\| `.` \| Match on forward strand \|
90	\| `,` \| Match on reverse strand \|
91	\| `ACGT` \| Mismatch (uppercase = forward) \|
92	\| `acgt` \| Mismatch (lowercase = reverse) \|
93	\| `^Q` \| Start of read (Q = MAPQ as ASCII) \|
94	\| `$` \| End of read \|
95	\| `+NNN` \| Insertion of N bases \|
96	\| `-NNN` \| Deletion of N bases \|
97	\| `*` \| Deleted base \|
98	\| `>` / `<` \| Reference skip (intron) \|
99
100	## Quality Filtering Options
101
102	### Minimum Mapping Quality
103	```bash
104	samtools mpileup -f reference.fa -q 20 input.bam
105	```
106
107	### Minimum Base Quality
108	```bash
109	samtools mpileup -f reference.fa -Q 20 input.bam
110	```
111
112	### Combined Quality Filters
113	```bash
114	samtools mpileup -f reference.fa -q 20 -Q 20 input.bam
115	```
116
117	### Maximum Depth (Critical Trap)
118	```bash
119	# samtools mpileup default -d 8000 silently truncates targeted / mt-DNA / amplicon / UMI-deduped data
120	# bcftools mpileup default -d 250 is far lower; both must be set explicitly when piping
121	samtools mpileup -f reference.fa -d 0 input.bam # no cap
122	samtools mpileup -f reference.fa -d 1000000 input.bam # explicit high cap
123
124	# WRONG -- samtools 8000 cap, then bcftools 250 cap re-applied
125	samtools mpileup -f ref.fa in.bam \| bcftools call -mv
126
127	# RIGHT -- single tool, explicit -d
128	bcftools mpileup -d 1000000 -f ref.fa in.bam \| bcftools call -mv
129	```
130
131	## BAQ: Base Alignment Quality (Critical Default)
132
133	When `-f ref.fa` is passed, BAQ is enabled by default. BAQ Phred-scales the probability that a base is misaligned (HMM realignment over a small window) and reduces base quality near indels. Tradeoffs: ~30% slower; suppresses FP SNVs

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6 picks

Type