Snippy 3.2 error

aworwui · March 9, 2018, 1:21am

Kindly help me resolve this error from snippy as part of nullarbor pipeline.
I’m running snippy 3.2 as part of nullarbor pipeline and the run terminate with this error below

[01:03:22] Patching variant sites into whole genome alignment…
Use of uninitialized value in substr at /home/linuxbrew/.linuxbrew/bin/snippy-core line 99.
substr outside of string at /home/linuxbrew/.linuxbrew/bin/snippy-core line 99.
Makefile:163870: recipe for target ‘core.aln’ failed
make: *** [core.aln] Error 255

regards
Archie

mattbull · March 9, 2018, 9:42am

Please could you show me snps.log from one of the samples?

aworwui · March 9, 2018, 9:48am

Below is the content of one of snsps.log file

cd /home/ubuntu/mrc_data/nullarbor_output

/home/linuxbrew/.linuxbrew/bin/snippy --cpus 4 --force --outdir ERR1065578/ERR1065578 --ref /home/ubuntu/mrc_data/ATCC_700669.fasta --R1 ERR1065578/R1.fq.gz --R2 ERR1065578/R2.fq.gz

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA… 0.03 sec
[bwa_index] Construct BWT for the packed sequence…
[bwa_index] 1.07 seconds elapse.
[bwa_index] Update BWT… 0.02 sec
[bwa_index] Pack forward-only FASTA… 0.02 sec
[bwa_index] Construct SA from BWT and Occ… 0.35 sec
[main] Version: 0.7.17-r1188
[main] CMD: bwa index reference/ref.fa
[main] Real time: 1.858 sec; CPU: 1.488 sec

mkdir reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

mkdir reference/ref && bgzip -c reference/ref.gff > reference/ref/genes.gff.gz

(bwa mem -v 2 -M -R ‘@RG\tID:snps\tSM:snps’ -t 4 reference/ref.fa /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/R1.fq.gz /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/R2.fq.gz | samtools view -u -T reference/ref.fa -F 3844 -q 60 | samtools sort --reference reference/ref.fa > snps.bam)

[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (295, 379, 455)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 775)
[M::mem_pestat] mean and std.dev: (342.65, 103.25)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 935)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (404, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (188, 728)
[M::mem_pestat] mean and std.dev: (460.12, 80.52)
[M::mem_pestat] low and high boundaries for proper pairs: (80, 836)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (404, 419, 423)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (366, 461)
[M::mem_pestat] mean and std.dev: (420.00, 9.89)
[M::mem_pestat] low and high boundaries for proper pairs: (347, 480)
[M::mem_pestat] analyzing insert size distribution for orientation RR…
[M::mem_pestat] (25, 50, 75) percentile: (6146, 6169, 6216)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (6006, 6356)
[M::mem_pestat] mean and std.dev: (6194.80, 41.85)
[M::mem_pestat] low and high boundaries for proper pairs: (5936, 6426)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (259, 314, 373)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (31, 601)
[M::mem_pestat] mean and std.dev: (318.22, 95.76)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 715)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 455, 514)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (187, 732)
[M::mem_pestat] mean and std.dev: (461.85, 80.51)
[M::mem_pestat] low and high boundaries for proper pairs: (78, 841)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (534, 588, 639)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (324, 849)
[M::mem_pestat] mean and std.dev: (582.38, 77.97)
[M::mem_pestat] low and high boundaries for proper pairs: (219, 954)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (300, 330, 432)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 696)
[M::mem_pestat] mean and std.dev: (344.19, 97.45)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 828)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (189, 729)
[M::mem_pestat] mean and std.dev: (461.72, 79.80)
[M::mem_pestat] low and high boundaries for proper pairs: (81, 837)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (259, 278, 486)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 940)
[M::mem_pestat] mean and std.dev: (345.53, 169.89)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 1167)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (403, 453, 511)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (187, 727)
[M::mem_pestat] mean and std.dev: (459.27, 80.26)
[M::mem_pestat] low and high boundaries for proper pairs: (79, 835)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (489, 546, 577)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (313, 753)
[M::mem_pestat] mean and std.dev: (528.20, 61.32)
[M::mem_pestat] low and high boundaries for proper pairs: (225, 841)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (280, 331, 390)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (60, 610)
[M::mem_pestat] mean and std.dev: (340.91, 64.82)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 720)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (191, 726)
[M::mem_pestat] mean and std.dev: (460.78, 79.33)
[M::mem_pestat] low and high boundaries for proper pairs: (84, 833)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (1293, 3843, 4105)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 9729)
[M::mem_pestat] mean and std.dev: (2964.50, 1410.47)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 12541)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 455, 514)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (187, 732)
[M::mem_pestat] mean and std.dev: (462.03, 79.99)
[M::mem_pestat] low and high boundaries for proper pairs: (78, 841)
[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: (1295, 3853, 4114)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 9752)
[M::mem_pestat] mean and std.dev: (2991.27, 1380.72)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 12571)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (286, 371, 439)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 745)
[M::mem_pestat] mean and std.dev: (354.16, 101.00)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 898)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (189, 729)
[M::mem_pestat] mean and std.dev: (460.60, 80.07)
[M::mem_pestat] low and high boundaries for proper pairs: (81, 837)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (213, 329, 399)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 771)
[M::mem_pestat] mean and std.dev: (312.17, 106.40)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 957)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (189, 729)
[M::mem_pestat] mean and std.dev: (460.02, 79.34)
[M::mem_pestat] low and high boundaries for proper pairs: (81, 837)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (302, 340, 426)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (54, 674)
[M::mem_pestat] mean and std.dev: (329.74, 102.08)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 798)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 455, 514)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (190, 730)
[M::mem_pestat] mean and std.dev: (461.64, 79.20)
[M::mem_pestat] low and high boundaries for proper pairs: (82, 838)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (2394, 2460, 2511)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (2160, 2745)
[M::mem_pestat] mean and std.dev: (2457.39, 82.16)
[M::mem_pestat] low and high boundaries for proper pairs: (2043, 2862)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (246, 337, 397)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 699)
[M::mem_pestat] mean and std.dev: (305.59, 118.44)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 850)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (191, 726)
[M::mem_pestat] mean and std.dev: (461.19, 79.22)
[M::mem_pestat] low and high boundaries for proper pairs: (84, 833)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (256, 338, 395)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 673)
[M::mem_pestat] mean and std.dev: (315.13, 126.98)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 823)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 455, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 727)
[M::mem_pestat] mean and std.dev: (461.65, 79.14)
[M::mem_pestat] low and high boundaries for proper pairs: (85, 834)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (225, 316, 416)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 798)
[M::mem_pestat] mean and std.dev: (314.53, 119.40)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 989)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (404, 453, 511)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (190, 725)
[M::mem_pestat] mean and std.dev: (460.12, 79.51)
[M::mem_pestat] low and high boundaries for proper pairs: (83, 832)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (268, 331, 388)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (28, 628)
[M::mem_pestat] mean and std.dev: (318.04, 118.28)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 791)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (191, 726)
[M::mem_pestat] mean and std.dev: (460.29, 78.98)
[M::mem_pestat] low and high boundaries for proper pairs: (84, 833)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (249, 331, 403)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 711)
[M::mem_pestat] mean and std.dev: (304.85, 97.76)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 865)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 455, 514)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (187, 732)
[M::mem_pestat] mean and std.dev: (460.70, 80.70)
[M::mem_pestat] low and high boundaries for proper pairs: (78, 841)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (782, 833, 877)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (592, 1067)
[M::mem_pestat] mean and std.dev: (825.89, 72.36)
[M::mem_pestat] low and high boundaries for proper pairs: (497, 1162)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (260, 318, 427)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 761)
[M::mem_pestat] mean and std.dev: (334.15, 129.19)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 928)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 455, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 727)
[M::mem_pestat] mean and std.dev: (462.34, 79.61)
[M::mem_pestat] low and high boundaries for proper pairs: (85, 834)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (322, 443, 1242)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 3082)
[M::mem_pestat] mean and std.dev: (783.94, 599.08)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 4002)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (194, 724)
[M::mem_pestat] mean and std.dev: (461.78, 79.37)
[M::mem_pestat] low and high boundaries for proper pairs: (88, 830)
[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: (1338, 1863, 4664)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 11316)
[M::mem_pestat] mean and std.dev: (2445.00, 1547.23)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 14642)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (288, 344, 436)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 732)
[M::mem_pestat] mean and std.dev: (346.88, 103.17)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 880)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 512)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (191, 726)
[M::mem_pestat] mean and std.dev: (461.51, 79.12)
[M::mem_pestat] low and high boundaries for proper pairs: (84, 833)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (243, 285, 340)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (49, 534)
[M::mem_pestat] mean and std.dev: (298.38, 86.46)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 644)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (403, 453, 511)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (187, 727)
[M::mem_pestat] mean and std.dev: (460.16, 79.97)
[M::mem_pestat] low and high boundaries for proper pairs: (79, 835)
[M::mem_pestat] analyzing insert size distribution for orientation RF…
[M::mem_pestat] (25, 50, 75) percentile: (964, 8413, 8422)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 23338)
[M::mem_pestat] mean and std.dev: (5640.66, 3728.33)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 30796)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (267, 337, 405)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 681)
[M::mem_pestat] mean and std.dev: (334.08, 108.71)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 819)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 455, 514)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (190, 730)
[M::mem_pestat] mean and std.dev: (461.81, 79.58)
[M::mem_pestat] low and high boundaries for proper pairs: (82, 838)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (286, 345, 411)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 661)
[M::mem_pestat] mean and std.dev: (344.71, 88.23)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 786)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (189, 729)
[M::mem_pestat] mean and std.dev: (461.20, 79.70)
[M::mem_pestat] low and high boundaries for proper pairs: (81, 837)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (280, 336, 399)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (42, 637)
[M::mem_pestat] mean and std.dev: (320.91, 113.24)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 774)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (406, 455, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 727)
[M::mem_pestat] mean and std.dev: (461.41, 78.89)
[M::mem_pestat] low and high boundaries for proper pairs: (85, 834)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (214, 359, 405)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 787)
[M::mem_pestat] mean and std.dev: (294.00, 112.59)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 978)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (404, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (186, 731)
[M::mem_pestat] mean and std.dev: (459.88, 80.28)
[M::mem_pestat] low and high boundaries for proper pairs: (77, 840)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (279, 342, 380)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (77, 582)
[M::mem_pestat] mean and std.dev: (333.59, 86.61)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 683)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (405, 454, 513)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (189, 729)
[M::mem_pestat] mean and std.dev: (461.14, 79.60)
[M::mem_pestat] low and high boundaries for proper pairs: (81, 837)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] analyzing insert size distribution for orientation FF…
[M::mem_pestat] (25, 50, 75) percentile: (283, 376, 395)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (59, 619)
[M::mem_pestat] mean and std.dev: (323.08, 107.83)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 754)
[M::mem_pestat] analyzing insert size distribution for orientation FR…
[M::mem_pestat] (25, 50, 75) percentile: (404, 453, 510)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (192, 722)
[M::mem_pestat] mean and std.dev: (459.75, 78.82)
[M::mem_pestat] low and high boundaries for proper pairs: (86, 828)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[main] Version: 0.7.17-r1188
[main] CMD: bwa mem -v 2 -M -R @RG\tID:snps\tSM:snps -t 4 reference/ref.fa /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/R1.fq.gz /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/R2.fq.gz
[main] Real time: 258.599 sec; CPU: 946.232 sec
[bam_sort_core] merging from 2 files and 1 in-memory blocks…

samtools index snps.bam

samtools depth -aa -q 20 snps.bam | bgzip > snps.depth.gz

tabix -s 1 -b 2 -e 2 snps.depth.gz

fasta_generate_regions.py reference/ref.fa.fai 141148 > reference/ref.txt

freebayes-parallel reference/ref.txt 4 -p 1 -q 20 -m 60 --min-coverage 10 -V -f reference/ref.fa snps.bam > snps.raw.vcf

/home/linuxbrew/.linuxbrew/Cellar/snippy/3.2/bin/snippy-vcf_filter --minqual 10 --mincov 10 --minfrac 0.9 snps.raw.vcf > snps.filt.vcf

Parsing: snps.raw.vcf
Types: complex=2359 ins=138 snp=13308 mnp=428 del=151
Passed 16384/16712 variants (98.04%)

cp snps.filt.vcf snps.vcf

bgzip -c snps.vcf > snps.vcf.gz

tabix -p vcf snps.vcf.gz

/home/linuxbrew/.linuxbrew/Cellar/snippy/3.2/bin/snippy-vcf_to_tab --gff reference/ref.gff --ref reference/ref.fa --vcf snps.vcf > snps.tab

Loading reference: reference/ref.fa
Loaded 1 sequences.
Loading features: reference/ref.gff
Parsing variants: snps.vcf
Converted 16384 SNPs to TAB format.

vcf-consensus snps.vcf.gz < reference/ref.fa > snps.consensus.fa

/home/linuxbrew/.linuxbrew/Cellar/snippy/3.2/bin/snippy-vcf_filter --subs snps.filt.vcf > snps.filt.subs.vcf

Parsing: snps.filt.vcf
Types: complex=2294 mnp=428 snp=13308
Passed 16030/16384 variants (97.84%)

bgzip -c snps.filt.subs.vcf > snps.filt.subs.vcf.gz

tabix -p vcf snps.filt.subs.vcf.gz

vcf-consensus snps.filt.subs.vcf.gz < reference/ref.fa > snps.consensus.subs.fa

mattbull · March 9, 2018, 11:38am

No problems there, so the problem must be after snippy.

snippy-core is very robust, and there are no reports of this problem elsewhere on the GitHub repo, so this suggests that it might be a problem with your input data.

Looking at the code for snippy-core, I wonder if this is to do with a mismatch between sequence names in the reference and variant files. Please could you show me:

grep ">" /home/ubuntu/mrc_data/nullarbor_output/ref.fa

head /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/ERR1065578/snps.tab

aworwui · March 9, 2018, 12:04pm

Below is the output:-
grep “>” /home/ubuntu/mrc_data/nullarbor_output/ref.fa

FM211187.1_ATCC_700669 FM211187

head /home/ubuntu/mrc_data/nullarbor_output/ERR1065578/ERR1065578/snps.tab
CHROM POS TYPE REF ALT EVIDENCE FTYPE STRAND NT_POS AA_POS EFFECT LOCUS_TAG GENE PRODUCT
ENA|FM211187|FM211187.1_ATCC_700669 126 snp G T T:530 G:0
ENA|FM211187|FM211187.1_ATCC_700669 524 snp G A A:537 G:0
ENA|FM211187|FM211187.1_ATCC_700669 845 snp A C C:469 A:0
ENA|FM211187|FM211187.1_ATCC_700669 1064 snp G A A:473 G:0
ENA|FM211187|FM211187.1_ATCC_700669 1805 snp G A A:522 G:0
ENA|FM211187|FM211187.1_ATCC_700669 1981 snp G A A:572 G:0
ENA|FM211187|FM211187.1_ATCC_700669 1997 snp G A A:569 G:0
ENA|FM211187|FM211187.1_ATCC_700669 2650 snp C T T:532 C:0
ENA|FM211187|FM211187.1_ATCC_700669 2943 snp T C C:507 T:0

mattbull · March 9, 2018, 12:22pm

Okay - that’s the problem I suspect. snippy-core is trying to find the sequence called:

ENA|FM211187|FM211187.1_ATCC_700669

in the reference file to patch variants into, but there is only one sequence in there, called:

FM211187.1_ATCC_700669 FM211187

which doesn’t match.

You have two options:

Edit ref.fa, so that the first line reads:

>ENA|FM211187|FM211187.1_ATCC_700669

and run the make command again. Hopefully this picks up where the run broke before.

Leave the sequence name as it is in ref.fa, remove all of the nullarbor output files, then run nullarbor.pl again to regenerate the Makefile and restart the analysis from the beginning.

aworwui · March 12, 2018, 11:54am

Hi Mat,
Thanks and changing the ID in the reference fasta correct the error.
However I notice another error coming from imagemagick, below isthe error messages
convert: memory allocation failed tree.svg' @ error/svg.c/ReadSVGImage/3108. convert: no images definedtree.gif’ @ error/convert.c/ConvertImageCommand/3275.
Makefile:176074: recipe for target ‘tree.gif’ failed
make: *** [tree.gif] Error 1

mattbull · March 12, 2018, 12:39pm

I think this might be a problem with recently updated imagemagick.

Please could you show me the output from:

brew list --versions | grep imagemagick

aworwui · March 12, 2018, 1:18pm

Dear Mat,
Below is the output:
imagemagick 7.0.7-25 7.0.7-5 7.0.7-24

mattbull · March 12, 2018, 1:22pm

Thanks - could you try switching to an older version of imagemagick with:

brew switch imagemagick 7.0.7-5

and seeing if this fixes the problem by running:

cd /home/ubuntu/mrc_data/nullarbor_output/
convert tree.svg tree.gif

and checking to see if you see the same error?

aworwui · March 12, 2018, 1:51pm

Hi Mattbul,
Thanks, switching the version works great. It resolves the issue
Thanks Mattbull

mattbull · March 19, 2018, 1:51pm

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