alignment-sorting

Name: alignment-sorting
Author: GPTomics

1.1kstars185forks

How to install alignment-sorting?

gptomics/bioskills/alignment-sorting

$npx -y skills add GPTomics/bioSkills --skill alignment-sorting

Installs into the current project.

Summary

Sort alignment files by coordinate or read name using samtools and pysam. Use when preparing BAM files for indexing, variant calling, or paired-end analysis.

Files · 1

View on GitHub

SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: 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	# Alignment Sorting
13
14	Sort alignment files by coordinate or read name using samtools and pysam.
15
16	"Sort a BAM file" -> Reorder reads by genomic coordinate (for indexing/variant calling) or by name (for paired-end processing).
17	- CLI: `samtools sort -o sorted.bam input.bam`
18	- Python: `pysam.sort('-o', 'sorted.bam', 'input.bam')`
19
20	## Sort Orders
21
22	\| Order \| Flag \| Use Case \|
23	\|-------\|------\|----------\|
24	\| Coordinate \| default \| Indexing, visualization, variant calling \|
25	\| Name \| `-n` \| Paired-end processing, fixmate, markdup \|
26	\| Tag \| `-t TAG` \| Sort by specific tag value \|
27
28	## samtools sort
29
30	### Sort by Coordinate (Default)
31	```bash
32	samtools sort -o sorted.bam input.bam
33	```
34
35	### Sort by Read Name
36	```bash
37	samtools sort -n -o namesorted.bam input.bam
38	```
39
40	### Multi-threaded Sorting
41	```bash
42	samtools sort -@ 8 -o sorted.bam input.bam
43	```
44
45	### Control Memory Usage
46	```bash
47	samtools sort -m 4G -@ 4 -o sorted.bam input.bam
48	```
49
50	### Set Temporary Directory
51	```bash
52	samtools sort -T /tmp/sort_tmp -o sorted.bam input.bam
53	```
54
55	### Specify Output Format
56	```bash
57	# Output as BAM (default)
58	samtools sort -O bam -o sorted.bam input.bam
59
60	# Output as CRAM
61	samtools sort -O cram --reference ref.fa -o sorted.cram input.bam
62	```
63
64	### Sort by Tag
65	```bash
66	# Sort by cell barcode (10x Genomics)
67	samtools sort -t CB -o sorted_by_barcode.bam input.bam
68	```
69
70	### Pipe from Aligner
71	```bash
72	bwa mem ref.fa reads.fq \| samtools sort -o aligned.bam
73	```
74
75	## samtools collate vs sort -n
76
77	\| Tool \| Algorithm \| Speed \| Memory \| Output guarantee \|
78	\|------\|-----------\|-------\|--------\|------------------\|
79	\| `sort -n` \| Full lexicographic sort by QNAME \| Slowest \| Spills to `-T` \| Strict total order by name \|
80	\| `collate` \| Hash-bucket grouping \| ~3-10x faster \| Bounded \| Mates adjacent; between-mate order undefined \|
81
82	Use `collate` when extracting paired FASTQ, re-aligning, or streaming through markdup. Use `sort -n` only when a tool requires true lexicographic name order (e.g. RSEM, Salmon alignment-mode).
83
84	```bash
85	# Fast paired FASTQ extraction
86	samtools collate -O -u in.bam tmp_prefix \| \
87	samtools fastq -1 R1.fq.gz -2 R2.fq.gz -0 /dev/null -s /dev/null -n -
88
89	# Markdup pre-processing (collate beats sort -n here)
90	samtools collate -O -u in.bam tmp_prefix \| \
91	samtools fixmate -m -u - - \| \
92	samtools sort -u - \| \
93	samtools markdup - out.bam
94	```
95
96	### Sort Order Required by Downstream Tool
97
98	\| Operation \| Required sort \|
99	\|-----------\|---------------\|
100	\| `samtools index` \| coordinate (hard requirement) \|
101	\| `samtools fixmate -m` \| name (or collate; needs mates adjacent) \|
102	\| `samtools markdup` \| coordinate (after fixmate) \|
103	\| GATK MarkDuplicatesSpark \| coordinate or queryname \|
104	\| `samtools mpileup` / `bcftools mpileup` \| coordinate \|
105	\| GATK HaplotypeCaller, Mutect2 \| coordinate \|
106	\| featureCounts / HTSeq \| coordinate or name (`-p` for paired) \|
107	\| umi_tools dedup \| coordinate (with index) \|
108	\| fgbio GroupReadsByUmi \| any order accepted (template-coordinate recommended to avoid an internal re-sort) \|
109	\| fgbio CallMolecularConsensusReads \| grouped by MI tag (consumes GroupReadsByUmi output) \|
110	\| Sniffles, cuteSV, Manta, Delly \| coordinate (need SA tags) \|
111	\| Salmon alignment-mode \| name \|
112	\| RSEM (with STAR `--quantMode TranscriptomeSAM`) \| name (hard requirement) \|
113
114	## Check Sort Order
115
116	### From Header
117	```bash
118	samtools view -H input.bam \| grep "^@HD"
119	# SO:coordinate = coordinate sorted
120	# SO:queryname = name sorted
121	# SO:unsorted = not sorted
122	```
123
124	### Verify Sorted
125	```bash
126	# Check if coordinate sorted (returns 0 if sorted). Reset the position tracker
127	# on each new contig, else the POS reset at every chromosome boundary of a
128	# correctly sorted multi-contig BAM would falsely report "unsorted".
129	samtools view input.bam \| awk '$3!=c {c=$3; prev=0} $4<prev {exit 1} {prev=$4}'
130	# Simpler and authoritative: trust the @HD SO: header shown above.
131	```
132
133	## pysam Python Alternative
134
135	### Sort with pysam
136	```python
137	import pysam
138
139	pysam.sort('-o', 'sorted.bam', 'input.bam')
140	```
141
142	### Sort by Name
143	```python
144	pysam.sort('-n', '-o', 'namesorted.bam', 'input.bam')
145	```
146
147	### Sort with Options
148	```python
149	pysam.sort('-@', '4', '-m', '2G', '-o', 'sorted.bam', 'input.bam')
150	```
151
152	### Avoid In-Python Sorting
153
154	Do not load BAM records into a list and call `sorted()`. `pysam.sort()` calls samtools'

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alignment-sorting

1.1kstars185forks

View on GitHub

How to install alignment-sorting?

gptomics/bioskills/alignment-sorting

$npx -y skills add GPTomics/bioSkills --skill alignment-sorting

Installs into the current project.

Summary

Sort alignment files by coordinate or read name using samtools and pysam. Use when preparing BAM files for indexing, variant calling, or paired-end analysis.

Files · 1

View on GitHub

SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: 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	# Alignment Sorting
13
14	Sort alignment files by coordinate or read name using samtools and pysam.
15
16	"Sort a BAM file" -> Reorder reads by genomic coordinate (for indexing/variant calling) or by name (for paired-end processing).
17	- CLI: `samtools sort -o sorted.bam input.bam`
18	- Python: `pysam.sort('-o', 'sorted.bam', 'input.bam')`
19
20	## Sort Orders
21
22	\| Order \| Flag \| Use Case \|
23	\|-------\|------\|----------\|
24	\| Coordinate \| default \| Indexing, visualization, variant calling \|
25	\| Name \| `-n` \| Paired-end processing, fixmate, markdup \|
26	\| Tag \| `-t TAG` \| Sort by specific tag value \|
27
28	## samtools sort
29
30	### Sort by Coordinate (Default)
31	```bash
32	samtools sort -o sorted.bam input.bam
33	```
34
35	### Sort by Read Name
36	```bash
37	samtools sort -n -o namesorted.bam input.bam
38	```
39
40	### Multi-threaded Sorting
41	```bash
42	samtools sort -@ 8 -o sorted.bam input.bam
43	```
44
45	### Control Memory Usage
46	```bash
47	samtools sort -m 4G -@ 4 -o sorted.bam input.bam
48	```
49
50	### Set Temporary Directory
51	```bash
52	samtools sort -T /tmp/sort_tmp -o sorted.bam input.bam
53	```
54
55	### Specify Output Format
56	```bash
57	# Output as BAM (default)
58	samtools sort -O bam -o sorted.bam input.bam
59
60	# Output as CRAM
61	samtools sort -O cram --reference ref.fa -o sorted.cram input.bam
62	```
63
64	### Sort by Tag
65	```bash
66	# Sort by cell barcode (10x Genomics)
67	samtools sort -t CB -o sorted_by_barcode.bam input.bam
68	```
69
70	### Pipe from Aligner
71	```bash
72	bwa mem ref.fa reads.fq \| samtools sort -o aligned.bam
73	```
74
75	## samtools collate vs sort -n
76
77	\| Tool \| Algorithm \| Speed \| Memory \| Output guarantee \|
78	\|------\|-----------\|-------\|--------\|------------------\|
79	\| `sort -n` \| Full lexicographic sort by QNAME \| Slowest \| Spills to `-T` \| Strict total order by name \|
80	\| `collate` \| Hash-bucket grouping \| ~3-10x faster \| Bounded \| Mates adjacent; between-mate order undefined \|
81
82	Use `collate` when extracting paired FASTQ, re-aligning, or streaming through markdup. Use `sort -n` only when a tool requires true lexicographic name order (e.g. RSEM, Salmon alignment-mode).
83
84	```bash
85	# Fast paired FASTQ extraction
86	samtools collate -O -u in.bam tmp_prefix \| \
87	samtools fastq -1 R1.fq.gz -2 R2.fq.gz -0 /dev/null -s /dev/null -n -
88
89	# Markdup pre-processing (collate beats sort -n here)
90	samtools collate -O -u in.bam tmp_prefix \| \
91	samtools fixmate -m -u - - \| \
92	samtools sort -u - \| \
93	samtools markdup - out.bam
94	```
95
96	### Sort Order Required by Downstream Tool
97
98	\| Operation \| Required sort \|
99	\|-----------\|---------------\|
100	\| `samtools index` \| coordinate (hard requirement) \|
101	\| `samtools fixmate -m` \| name (or collate; needs mates adjacent) \|
102	\| `samtools markdup` \| coordinate (after fixmate) \|
103	\| GATK MarkDuplicatesSpark \| coordinate or queryname \|
104	\| `samtools mpileup` / `bcftools mpileup` \| coordinate \|
105	\| GATK HaplotypeCaller, Mutect2 \| coordinate \|
106	\| featureCounts / HTSeq \| coordinate or name (`-p` for paired) \|
107	\| umi_tools dedup \| coordinate (with index) \|
108	\| fgbio GroupReadsByUmi \| any order accepted (template-coordinate recommended to avoid an internal re-sort) \|
109	\| fgbio CallMolecularConsensusReads \| grouped by MI tag (consumes GroupReadsByUmi output) \|
110	\| Sniffles, cuteSV, Manta, Delly \| coordinate (need SA tags) \|
111	\| Salmon alignment-mode \| name \|
112	\| RSEM (with STAR `--quantMode TranscriptomeSAM`) \| name (hard requirement) \|
113
114	## Check Sort Order
115
116	### From Header
117	```bash
118	samtools view -H input.bam \| grep "^@HD"
119	# SO:coordinate = coordinate sorted
120	# SO:queryname = name sorted
121	# SO:unsorted = not sorted
122	```
123
124	### Verify Sorted
125	```bash
126	# Check if coordinate sorted (returns 0 if sorted). Reset the position tracker
127	# on each new contig, else the POS reset at every chromosome boundary of a
128	# correctly sorted multi-contig BAM would falsely report "unsorted".
129	samtools view input.bam \| awk '$3!=c {c=$3; prev=0} $4<prev {exit 1} {prev=$4}'
130	# Simpler and authoritative: trust the @HD SO: header shown above.
131	```
132
133	## pysam Python Alternative
134
135	### Sort with pysam
136	```python
137	import pysam
138
139	pysam.sort('-o', 'sorted.bam', 'input.bam')
140	```
141
142	### Sort by Name
143	```python
144	pysam.sort('-n', '-o', 'namesorted.bam', 'input.bam')
145	```
146
147	### Sort with Options
148	```python
149	pysam.sort('-@', '4', '-m', '2G', '-o', 'sorted.bam', 'input.bam')
150	```
151
152	### Avoid In-Python Sorting
153
154	Do not load BAM records into a list and call `sorted()`. `pysam.sort()` calls samtools'

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6 picks

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