Issue with chr name format

[mgandal]

I'm using sorted BAM files that have numeric chromosome names (no "chr" prefix). There seems to be an issue with quantification. If I remove "chr" from your degradation BED file chromosome names, I get 0's for quantification of regions on chromosomes 10-19.

If I sort the BED file so that numeric chromosomes are in numeric order (to match BAM files), I get the following error I think from wiggleTools:
"Position 10:714133 is before 9:140899137"
even though the BED file looks like

9	140049063	140050870	.	0	.
9	140791269	140791603	.	0	.
9	140833366	140833967	.	0	.
9	140899136	140899908	.	0	.
10	714132	714671	.	0	.
10	7830132	7830226	.	0	.
10	51565182	51565296	.	0	.
10	51580555	51580696	.	0	.
10	51632928	51633033	.	0	.
10	60028911	60029064	.	0	.

[nellore]

Hey! Are you sure you're sorting the bed file so numeric chromosomes are in numeric order? If 10:714133 < 9:1408991937, that may not be true. Are you doing
sort -k1,1n -k2,2n -k3,3n regions.bed?

Not sure that would solve your problem though. For the case where you just remove chr from the degradation bed, can you try running wiggletools directly using

wiggletools write_bg - trim regions.bed indexed_alignments.bam

and checking whether the output gives 0 coverage for regions chromosome 10 and up? Let me know what happens.

[mgandal]

Hi, Thanks. I think it is actually a problem with wiggletools.

Here is an example using wiggletools write_bg - trim regions.bed indexed_alignments.bam with a BAM file with numeric chromosomes. The BAM file is indexed and coordinate sorted:

@HD	VN:1.4	SO:coordinate
@SQ	SN:1	LN:249250621	UR:file:/RefGenome/genome.fa	M5:1b22b98cdeb4a9304cb5d48026a85128
@SQ	SN:2	LN:243199373	UR:file:/RefGenome/genome.fa	M5:a0d9851da00400dec1098a9255ac712e
@SQ	SN:3	LN:198022430	UR:file:/RefGenome/genome.fa	M5:fdfd811849cc2fadebc929bb925902e5
@SQ	SN:4	LN:191154276	UR:file:/RefGenome/genome.fa	M5:23dccd106897542ad87d2765d28a19a1
@SQ	SN:5	LN:180915260	UR:file:/RefGenome/genome.fa	M5:0740173db9ffd264d728f32784845cd7
@SQ	SN:6	LN:171115067	UR:file:/RefGenome/genome.fa	M5:1d3a93a248d92a729ee764823acbbc6b
@SQ	SN:7	LN:159138663	UR:file:/RefGenome/genome.fa	M5:618366e953d6aaad97dbe4777c29375e
@SQ	SN:8	LN:146364022	UR:file:/RefGenome/genome.fa	M5:96f514a9929e410c6651697bded59aec
@SQ	SN:9	LN:141213431	UR:file:/RefGenome/genome.fa	M5:3e273117f15e0a400f01055d9f393768
@SQ	SN:10	LN:135534747	UR:file:/RefGenome/genome.fa	M5:988c28e000e84c26d552359af1ea2e1d
@SQ	SN:11	LN:135006516	UR:file:/RefGenome/genome.fa	M5:98c59049a2df285c76ffb1c6db8f8b96
@SQ	SN:12	LN:133851895	UR:file:/RefGenome/genome.fa	M5:51851ac0e1a115847ad36449b0015864
@SQ	SN:13	LN:115169878	UR:file:/RefGenome/genome.fa	M5:283f8d7892baa81b510a015719ca7b0b
@SQ	SN:14	LN:107349540	UR:file:/RefGenome/genome.fa	M5:98f3cae32b2a2e9524bc19813927542e
@SQ	SN:15	LN:102531392	UR:file:/RefGenome/genome.fa	M5:e5645a794a8238215b2cd77acb95a078
@SQ	SN:16	LN:90354753	UR:file:/RefGenome/genome.fa	M5:fc9b1a7b42b97a864f56b348b06095e6
@SQ	SN:17	LN:81195210	UR:file:/RefGenome/genome.fa	M5:351f64d4f4f9ddd45b35336ad97aa6de
@SQ	SN:18	LN:78077248	UR:file:/RefGenome/genome.fa	M5:b15d4b2d29dde9d3e4f93d1d0f2cbc9c
@SQ	SN:19	LN:59128983	UR:file:/RefGenome/genome.fa	M5:1aacd71f30db8e561810913e0b72636d
@SQ	SN:20	LN:63025520	UR:file:/RefGenome/genome.fa	M5:0dec9660ec1efaaf33281c0d5ea2560f
@SQ	SN:21	LN:48129895	UR:file:/RefGenome/genome.fa	M5:2979a6085bfe28e3ad6f552f361ed74d
@SQ	SN:22	LN:51304566	UR:file:/RefGenome/genome.fa	M5:a718acaa6135fdca8357d5bfe94211dd
@SQ	SN:X	LN:155270560	UR:file:/RefGenome/genome.fa	M5:7e0e2e580297b7764e31dbc80c2540dd
@SQ	SN:Y	LN:59373566	UR:file:/RefGenome/genome.fa	M5:1fa3474750af0948bdf97d5a0ee52e51

For simplicity, I made a BED file with only one region per chromosome. If I sort the BED file as you suggest sort -k1,1n -k2,2n -k3,3n regions_noCh_v1.bed, and run the wiggletools command wiggletools write_bg - trim regions.bed indexed_alignments.bam I get the following error:

Position 10:714133 is before 9:32552799

even though the BED file is corrected sorted by chromosome in numeric order:

1	6257711	6257893	.	0	.
2	1872090	1872390	.	0	.
3	197682531	197683078	.	0	.
4	602915	603654	.	0	.
5	176279272	176279378	.	0	.
6	16430725	16431049	.	0	.
6	166265710	166267024	.	0	.
7	713588	713686	.	0	.
8	121457307	121457378	.	0	.
9	32552798	32552977	.	0	.
10	714132	714671	.	0	.
11	14520383	14520556	.	0	.
12	6601303	6601633	.	0	.
13	114561069	114562803	.	0	.
14	23495058	23495584	.	0	.
15	101812398	101813061	.	0	.
16	648960	649176	.	0	.
17	79857795	79858128	.	0	.
18	3253886	3254048	.	0	.
19	58906048	58906170	.	0	.
20	60963364	60963575	.	0	.
21	35286753	35286804	.	0	.

If instead I use the BED file sorted as it was previously (as if the chr prefix was still present) wiggletools runs but only returns outputs for chromosomes 1-9

1	6257711	6257893	.	0	.
10	714132	714671	.	0	.
11	14520383	14520556	.	0	.
12	6601303	6601633	.	0	.
13	114561069	114562803	.	0	.
14	23495058	23495584	.	0	.
15	101812398	101813061	.	0	.
16	648960	649176	.	0	.
17	79857795	79858128	.	0	.
18	3253886	3254048	.	0	.
19	58906048	58906170	.	0	.
2	1872090	1872390	.	0	.
20	60963364	60963575	.	0	.
21	35286753	35286804	.	0	.
3	197682531	197683078	.	0	.
4	602915	603654	.	0	.
5	176279272	176279378	.	0	.
6	16430725	16431049	.	0	.
6	166265710	166267024	.	0	.
7	713588	713686	.	0	.
8	121457307	121457378	.	0	.
9	32552798	32552977	.	0	.

[mgandal]

Yes, looks like the wiggletools BAM parser is having issues
Ensembl/WiggleTools#19 (comment)

[nellore]

Glad this has been figured out! Hope it's resolved soon.