ale_score.sh error - tmp file "doesn't look like a PPM file"

[Dwrgi]

Hi, I've tried to generate an ale score for my assembly using the ale_score.sh script, but I get this error message and I can't figure out what's going wrong and where:

Error message:

[main] CMD: bwa mem -t 4 /tmp/tmp.CgDerxPFDu filtered_reads/Pol_01_illumina_1.fastq.gz filtered_reads/Pol_01_illumina_2.fastq.gz [main] Real time: 88.395 sec; CPU: 336.350 sec Output file will be '/tmp/tmp.QPXOcKrq0l'.
| Loading Image (caching)

*** '/tmp/tmp.BB5XLQvG32' doesn't look like a PPM file.

(To handle other file types, use a version of ALE with ImageMagick support enabled.)

I've got ALE version 0.9.0.3 as the most up to date I can get via conda.

Command and full output:

$ bash ale_score.sh trycycler_medaka_polypolish_polca.fasta filtered_reads/Pol_01_illumina_1.fastq.gz filtered_reads/Pol_01_illumina_2.fastq.gz 4 [bwa_index] Pack FASTA... 0.07 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 2.29 seconds elapse. [bwa_index] Update BWT... 0.09 sec [bwa_index] Pack forward-only FASTA... 0.04 sec [bwa_index] Construct SA from BWT and Occ... 0.53 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index -p /tmp/tmp.CgDerxPFDu trycycler_medaka_polypolish_polca.fasta [main] Real time: 3.099 sec; CPU: 3.022 sec [M::bwa_idx_load_from_disk] read 0 ALT contigs [M::process] read 176862 sequences (40000391 bp)... [M::process] read 178286 sequences (40000004 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (40, 84881, 2, 35) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (506, 591, 753) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (12, 1247) [M::mem_pestat] mean and std.dev: (571.33, 176.03) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1494) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (624, 722, 840) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 1272) [M::mem_pestat] mean and std.dev: (738.03, 162.57) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1488) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (493, 570, 800) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1414) [M::mem_pestat] mean and std.dev: (587.12, 222.09) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1721) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176862 reads in 39.133 CPU sec, 10.776 real sec [M::process] read 174134 sequences (40000054 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (25, 85500, 0, 34) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (401, 601, 761) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1481) [M::mem_pestat] mean and std.dev: (584.83, 254.54) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1841) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (612, 707, 818) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (200, 1230) [M::mem_pestat] mean and std.dev: (720.58, 155.29) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1436) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (541, 614, 745) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (133, 1153) [M::mem_pestat] mean and std.dev: (577.10, 162.33) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1357) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 178286 reads in 37.448 CPU sec, 9.515 real sec [M::process] read 176118 sequences (40000172 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (45, 83635, 1, 30) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (504, 625, 792) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1368) [M::mem_pestat] mean and std.dev: (621.63, 222.08) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1656) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (641, 747, 879) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (165, 1355) [M::mem_pestat] mean and std.dev: (767.72, 179.42) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1593) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (515, 656, 832) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1466) [M::mem_pestat] mean and std.dev: (631.14, 209.05) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1783) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 174134 reads in 39.470 CPU sec, 10.042 real sec [M::process] read 176224 sequences (40000290 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (49, 84521, 0, 31) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (457, 552, 663) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (45, 1075) [M::mem_pestat] mean and std.dev: (524.67, 183.77) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1281) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (625, 723, 846) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (183, 1288) [M::mem_pestat] mean and std.dev: (741.70, 166.53) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1509) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (594, 706, 879) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (24, 1449) [M::mem_pestat] mean and std.dev: (750.83, 226.62) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1734) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176118 reads in 41.137 CPU sec, 10.416 real sec [M::process] read 180900 sequences (40000090 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (34, 84623, 1, 46) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (493, 627, 841) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1537) [M::mem_pestat] mean and std.dev: (657.34, 285.27) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1885) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (620, 717, 835) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (190, 1265) [M::mem_pestat] mean and std.dev: (733.27, 162.55) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1480) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (490, 573, 681) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (108, 1063) [M::mem_pestat] mean and std.dev: (566.50, 150.27) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1254) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176224 reads in 42.410 CPU sec, 10.729 real sec [M::process] read 176218 sequences (40000135 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (31, 86626, 0, 43) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (469, 578, 662) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (83, 1048) [M::mem_pestat] mean and std.dev: (553.00, 150.07) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1241) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (590, 677, 775) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (220, 1145) [M::mem_pestat] mean and std.dev: (687.70, 139.87) [M::mem_pestat] low and high boundaries for proper pairs: (35, 1330) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (429, 521, 647) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1083) [M::mem_pestat] mean and std.dev: (523.48, 190.70) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1301) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 180900 reads in 39.740 CPU sec, 10.198 real sec [M::process] read 178148 sequences (40000234 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (32, 84408, 1, 37) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (542, 643, 871) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1529) [M::mem_pestat] mean and std.dev: (597.45, 227.31) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1858) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (611, 705, 815) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (203, 1223) [M::mem_pestat] mean and std.dev: (718.85, 154.51) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1427) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (441, 536, 669) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1125) [M::mem_pestat] mean and std.dev: (532.14, 149.13) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1353) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176218 reads in 39.621 CPU sec, 10.075 real sec [M::process] read 70084 sequences (15970297 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (36, 85400, 0, 26) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (454, 535, 712) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1228) [M::mem_pestat] mean and std.dev: (576.29, 170.59) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1486) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (606, 700, 808) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (202, 1212) [M::mem_pestat] mean and std.dev: (713.48, 152.93) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1414) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (444, 575, 684) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1164) [M::mem_pestat] mean and std.dev: (573.40, 181.24) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1404) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 178148 reads in 39.447 CPU sec, 10.011 real sec [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (12, 33640, 0, 14) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (585, 711, 838) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (79, 1344) [M::mem_pestat] mean and std.dev: (716.17, 243.83) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1691) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (622, 719, 834) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (198, 1258) [M::mem_pestat] mean and std.dev: (734.26, 161.02) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1470) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (483, 567, 741) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1257) [M::mem_pestat] mean and std.dev: (575.46, 124.30) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1515) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 70084 reads in 16.092 CPU sec, 4.086 real sec [main] Version: 0.7.17-r1188 [main] CMD: bwa mem -t 4 /tmp/tmp.CgDerxPFDu filtered_reads/Pol_01_illumina_1.fastq.gz filtered_reads/Pol_01_illumina_2.fastq.gz [main] Real time: 88.395 sec; CPU: 336.350 sec Output file will be '/tmp/tmp.QPXOcKrq0l'.
| Loading Image (caching)

*** '/tmp/tmp.BB5XLQvG32' doesn't look like a PPM file.

(To handle other file types, use a version of ALE with ImageMagick support enabled.)

[rrwick]

Based on that version number, I suspect you've got the wrong ALE tool installed. There are a few tools named 'ALE', but the one you want is this one: https://github.com/sc932/ALE. The correct ALE is in bioconda, but the ALE in conda-forge is something else. ALE is also pretty easy to build from source (just clone it and run make) so you can skip conda altogether if that makes things easier.

Hope that helps!

[Dwrgi]

Hi, Thanks! I've chucked out the ale that was there and installed the correct one from github, but I still get an image/PPM related error:

bash ale_score.sh Pol_01_trycycler_medaka_polypolish_polca.fasta filtered_reads/Pol_01_illumina_1.fastq.gz filtered_reads/Pol_01_illumina_2.fastq.gz 4 [bwa_index] Pack FASTA... 0.08 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 2.10 seconds elapse. [bwa_index] Update BWT... 0.09 sec [bwa_index] Pack forward-only FASTA... 0.04 sec [bwa_index] Construct SA from BWT and Occ... 0.70 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index -p /tmp/tmp.xD5Xz1l7Sg Pol_01_trycycler_medaka_polypolish_polca.fasta [main] Real time: 3.109 sec; CPU: 3.021 sec [M::bwa_idx_load_from_disk] read 0 ALT contigs [M::process] read 176862 sequences (40000391 bp)... [M::process] read 178286 sequences (40000004 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (40, 84881, 2, 35) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (506, 591, 753) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (12, 1247) [M::mem_pestat] mean and std.dev: (571.33, 176.03) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1494) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (624, 722, 840) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 1272) [M::mem_pestat] mean and std.dev: (738.03, 162.57) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1488) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (493, 570, 800) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1414) [M::mem_pestat] mean and std.dev: (587.12, 222.09) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1721) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176862 reads in 36.719 CPU sec, 9.351 real sec [M::process] read 174134 sequences (40000054 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (25, 85500, 0, 34) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (401, 601, 761) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1481) [M::mem_pestat] mean and std.dev: (584.83, 254.54) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1841) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (612, 707, 818) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (200, 1230) [M::mem_pestat] mean and std.dev: (720.58, 155.29) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1436) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (541, 614, 745) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (133, 1153) [M::mem_pestat] mean and std.dev: (577.10, 162.33) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1357) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 178286 reads in 37.160 CPU sec, 9.622 real sec [M::process] read 176118 sequences (40000172 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (45, 83635, 1, 30) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (504, 625, 792) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1368) [M::mem_pestat] mean and std.dev: (621.63, 222.08) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1656) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (641, 747, 879) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (165, 1355) [M::mem_pestat] mean and std.dev: (767.72, 179.42) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1593) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (515, 656, 832) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1466) [M::mem_pestat] mean and std.dev: (631.14, 209.05) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1783) [M::mem_pestat] skip orientation FF

[M::mem_pestat] skip orientation RR

[M::mem_process_seqs] Processed 174134 reads in 36.342 CPU sec, 9.517 real sec

[M::process] read 176224 sequences (40000290 bp)...

[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (49, 84521, 0, 31)

[M::mem_pestat] analyzing insert size distribution for orientation FF...

[M::mem_pestat] (25, 50, 75) percentile: (457, 552, 663) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (45, 1075) [M::mem_pestat] mean and std.dev: (524.67, 183.77) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1281) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (625, 723, 846) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (183, 1288) [M::mem_pestat] mean and std.dev: (741.70, 166.53) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1509) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (594, 706, 879) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (24, 1449)

[M::mem_pestat] mean and std.dev: (750.83, 226.62)

[M::mem_pestat] low and high boundaries for proper pairs: (1, 1734)

[M::mem_pestat] skip orientation FF

[M::mem_pestat] skip orientation RR

[M::mem_process_seqs] Processed 176118 reads in 37.129 CPU sec, 9.470 real sec [M::process] read 180900 sequences (40000090 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (34, 84623, 1, 46) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (493, 627, 841) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1537) [M::mem_pestat] mean and std.dev: (657.34, 285.27) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1885) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (620, 717, 835) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (190, 1265) [M::mem_pestat] mean and std.dev: (733.27, 162.55) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1480) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (490, 573, 681) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (108, 1063) [M::mem_pestat] mean and std.dev: (566.50, 150.27) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1254) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176224 reads in 37.580 CPU sec, 9.506 real sec [M::process] read 176218 sequences (40000135 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (31, 86626, 0, 43) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (469, 578, 662) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (83, 1048) [M::mem_pestat] mean and std.dev: (553.00, 150.07) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1241) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (590, 677, 775) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (220, 1145) [M::mem_pestat] mean and std.dev: (687.70, 139.87) [M::mem_pestat] low and high boundaries for proper pairs: (35, 1330) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (429, 521, 647) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1083) [M::mem_pestat] mean and std.dev: (523.48, 190.70) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1301) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 180900 reads in 40.264 CPU sec, 10.208 real sec [M::process] read 178148 sequences (40000234 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (32, 84408, 1, 37) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (542, 643, 871) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1529) [M::mem_pestat] mean and std.dev: (597.45, 227.31) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1858) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (611, 705, 815) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (203, 1223) [M::mem_pestat] mean and std.dev: (718.85, 154.51) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1427) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (441, 536, 669) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1125) [M::mem_pestat] mean and std.dev: (532.14, 149.13) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1353) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 176218 reads in 37.520 CPU sec, 9.717 real sec [M::process] read 70084 sequences (15970297 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (36, 85400, 0, 26) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (454, 535, 712) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1228) [M::mem_pestat] mean and std.dev: (576.29, 170.59) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1486) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (606, 700, 808) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (202, 1212) [M::mem_pestat] mean and std.dev: (713.48, 152.93) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1414) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (444, 575, 684) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1164) [M::mem_pestat] mean and std.dev: (573.40, 181.24) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1404) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 178148 reads in 36.387 CPU sec, 9.223 real sec [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (12, 33640, 0, 14) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (585, 711, 838) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (79, 1344) [M::mem_pestat] mean and std.dev: (716.17, 243.83) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1691) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (622, 719, 834) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (198, 1258) [M::mem_pestat] mean and std.dev: (734.26, 161.02) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1470) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (483, 567, 741) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1257) [M::mem_pestat] mean and std.dev: (575.46, 124.30) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1515) [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RR [M::mem_process_seqs] Processed 70084 reads in 13.951 CPU sec, 3.547 real sec [main] Version: 0.7.17-r1188 [main] CMD: bwa mem -t 4 /tmp/tmp.xD5Xz1l7Sg filtered_reads/Pol_01_illumina_1.fastq.gz filtered_reads/Pol_01_illumina_2.fastq.gz [main] Real time: 82.574 sec; CPU: 314.927 sec Output file will be '/tmp/tmp.YtrOWZUnlp'.
| Loading Image (caching)

*** '/tmp/tmp.EgnoFiruIa' doesn't look like a PPM file.

(To handle other file types, use a version of ALE with ImageMagick support enabled.)

ALE -h Welcome to the Assembly Likelihood Estimator! (C) 2010 Scott Clark

Usage: ALE [-options] alignments.[s|b]am assembly.fasta[.gz] ALEoutput.txt

Options: nt loat tring [default] -h or --help : print out this help --kmer : Kmer depth for kmer stats [4] --qOff : Quality ascii offset (illumina) [33] or 64 (or 0) --pl : placementOutputBAM --pm : library parameter file (auto outputs .param) --nout : only output meta information (no per base) [off] --minLL : the minimum log Likelihood (-120) --metagenome : Evaluate each contig independently for depth & kmer metrics --realign[=matchScore,misMatchPenalty,gapOpenPenalty,gapExtPenalty,minimumSoftClip (default: 1,3,11,4,8) ] Realign reads with Striped-Smith-Waterman honoring ambiguous reference bases and stacking homo-polymer indels for PacBio, try --realign=1,5,2,1,20 (similar to BWA-SW recommendations) --SNPreport : Creates a new text file reporting all SNP phasing observed by a read against ambiguous bases in the reference --minQual : Minimum quality score to use in Z-normalization (default 3). Illumina quality scores can be unreliable below this threshold

[rrwick]

That error message (use a version of ALE with ImageMagick support enabled) suggests to me that you have a different ALE installed: Anti-Lamenessing Engine (I know nothing about it - just found in a Google search). But you also clearly have the correct ALE installed, based on the help output.

So I think the issue might be with your environment, PATH variable or something like that. Maybe try to find all executables you have named ALE and uninstall the image-processing one?

[Dwrgi]

Thanks! Now that I knew what I was looking for I managed to find it (only the 'wrong' ale (ale 0.9.0.3-5) showed up in the list of installed packages) and get rid of it, so everything is working fine. :)

[rrwick]

Great - glad to hear it!