myassembly.myreads.as0.65.nm5.molecule.tsv in null

[shameem356]

Hello,

I am running tigmint with the following command.But myassembly.myreads.as0.65.nm5.molecule.tsv file is coming as null.Please can let me know the reason ?

tigmint-make tigmint draft=myassembly reads=myreads t=16

[lcoombe]

Hello, Could you provide the list of files in your working directory (ls -lah), and the log produced by tigmint? Were there any errors generated?

[shameem356]

Below are the list of files from working directory.There were no error messages generated. myassembly.fa myassembly.fa.amb myassembly.fa.ann myassembly.fa.bwt myassembly.fa.fai myassembly.fa.pac myassembly.fa.sa myassembly.myreads.as0.65.nm5.molecule.bed myassembly.myreads.as0.65.nm5.molecule.size2000.bed myassembly.myreads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa myassembly.myreads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa.bed myassembly.myreads.as0.65.nm5.molecule.tsv myassembly.myreads.bam myassembly.myreads.sam myassembly.myreads.sortbx.bam myassembly.tigmint.fa myreads.fq.gz nohup.out old_nohup.sh out.txt run_bwa.sh run_tigmint.sh

These are the tail part of log file.

/data/labs/genomics_lab/shared/shameem/apps/tigmint/bin/tigmint-molecule -b myassembly.myreads.sortbx.bam -o myassembly.myreads.as0.65.nm5.molecule.tsv -a 0.65 -n 5 -q 0 -d 50000 awk 'NR>1 { print $1"\t"$2-1"\t"$3-1"\tReads="$7",Size="$4",Mapq="$8",AS="$9",NM="$10",BX="$5",MI="$6"\t"$7 }' myassembly.myreads.as0.65.nm5.molecule.tsv \ | sort -k1,1 -k2,2n -k3,3n >myassembly.myreads.as0.65.nm5.molecule.bed awk '$3 - $2 >= 2000' myassembly.myreads.as0.65.nm5.molecule.bed >myassembly.myreads.as0.65.nm5.molecule.size2000.bed samtools faidx myassembly.fa /data/labs/genomics_lab/shared/shameem/apps/tigmint/bin/tigmint-cut -p16 -w1000 -n20 -t0 -o myassembly.myreads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa myassembly.fa myassembly.myreads.as0.65.nm5.molecule.size2000.bed Started at: 2018-05-17 16:12:50.703750 Reading contig lengths... Finding breakpoints... Cutting assembly at breakpoints... DONE! Ended at: 2018-05-17 16:12:57.061767 ln -sf myassembly.myreads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa myassembly.tigmint.fa

[lcoombe]

Ok thanks -- can you also provide a few lines of the myassembly.myreads.sortbx.bam file? (Just with samtools view)

[shameem356]

K00154:45:HKCNYBBXX:8:2210:30076:13025_TCACGCTAGGACAGCT 99 1 33 27 13M3I61M = 652 716 TCATATCATATAATATTATATTAATAATATTCATTGTAATATATTATTATAGAAGATATCCACAATTTTACGGCAAT JFFF7FFFJJJ-FJAFJJA<JJJFJAAFFJJJFJJFJ<7F<-AJJJJFJF<FJF<<AFFFJ7AAJJJJJFJF7FJ<< NM:i:4 MD:Z:11C62 MC:Z:3S97M AS:i:62 XS:i:72 XA:Z:7263,-76685,77M,1; K00154:45:HKCNYBBXX:4:2115:13220:48175_CTCTTTCAGAAGCCGT 99 1 53 0 77M = 357 390 ATAGTATTAATTGTAATATATTATTATAGAAGATATCCACAATTTTACGGCAATAATAATATTTGTGTTACTAAATA JFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJFJJ-FJJJJJJJJFJAJAF NM:i:2 MD:Z:3A4C68 MC:Z:86M14S AS:i:68 XS:i:68 XA:Z:7263,-76662,77M,2; K00154:45:HKCNYBBXX:8:2102:15899:2070_AAGGGTGGTGAACCTT 113 1 57 0 41M36S 7263 76920 0 TATTCATTGTAATATATTATTATAGAAGATATCCACAATTTGATATAGTGAAAATTATAGCGACATTCCTCCACTTT JJJJJJJJJJJJJJJJJFJFJJAFJJJJJJJJJJF<FJJJFJJJJJJJFJJJJJJFJJJJJJJJJFFJJFJJJJJJJ NM:i:0 MD:Z:41 MC:Z:75M25S AS:i:41 XS:i:41 SA:Z:7263,76772,-,38S39M,0,0; XA:Z:7263,+76694,36S41M,0; K00154:45:HKCNYBBXX:8:1206:14336:23909_GATGAAAAGGTCAATA 99 1 72 40 68M1D9M = 228 253 ATTATTATAGAAGATATCCACAATTTTACGGCAATAATAATATTTGTGTTACTAAATATGTAGTACATATTATTTTG JJJFJJJJJJJFJJFJFJJJJJJJJJJJJJ7AJJJJJJJJJJFFJJF<FAJJJJJJJJJFJJJJJJJJJJJJJJJJJ NM:i:1 MD:Z:68^A9 MC:Z:27M1I3M1I20M1I47M AS:i:70 XS:i:72 XA:Z:7263,-76643,77M,1; K00154:45:HKCNYBBXX:8:2210:24058:17860_TGTTCGCTCTCATGTT 163 1 132 33 11S8M1D81M = 372 317 CGCTCTCATGTTAGTACATATTATTTTGAAAATAATATTTTATAATATATTATTGATAAAGGATATTAGAATATTCTCCGTGTATTAGGGATTGGACATG AAFFFFJJJJJJJJJJJJJJJJJJJJAJJJJJJJJJJJJJJJJJJJJJJJJJJJAFFJJJJFFJJJJJJJJJJAFJFFFFFJFJJFJJ<7AFAFJFJJ<J NM:i:1 MD:Z:8^A81 MC:Z:77M AS:i:82 XS:i:76 XA:Z:7263,-76569,30M2D59M11S,3; K00154:45:HKCNYBBXX:8:2107:18213:6290_CTACCTGAGTCATGCT 163 1 143 60 100M = 412 346 TATTTTGAAAATAATATTTTATAATATATTATTGATAAAGGATATTAGAATATTCTCCGTGTATTAGGGATTGGACATGTTTTTGAATCCATTTTTCGCT AAAFFJJJJJJJJJJJJJJJJJJJJJFFFJJJJJFJJJFJJJJJJJJJJJJJJJJJJFJJJJJJJJJAAJJFJ-AJ<JFFJFJJFJJF-AFJF-AFJ<F< NM:i:0 MD:Z:100 MC:Z:77M AS:i:100 XS:i:89 XA:Z:7263,-76551,3S48M2D49M,2; K00154:45:HKCNYBBXX:8:2107:18254:6290_CTACCTGAGTCATGCT 163 1 143 60 100M = 412 346 TATTTTGAAAATAATATTTTATAATATATTATTGATAAAGGATATTAGAATATTCTCCGTGTATTAGGGATTGGACATGTTTTTGAATCCATTTTTCGCT AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJAFJJJFAJJJJFFAFFJAJAJAFFFFAAJJFJ< NM:i:0 MD:Z:100 MC:Z:77M AS:i:100 XS:i:89 XA:Z:7263,-76551,3S48M2D49M,2;

[shameem356]

By mistake I closed the ticket.Please can you open it.

[lcoombe]

It looks like you don't have the barcode in the BX tag in those alignments. Tigmint expects the chromium barcode to be in the BX:Z tag of the alignments, and will sort the alignment file by this tag. Make sure your chromium reads have the "BX:Z" tag in the comment of the read header: Ex:

@E00316:145:HMVG3CCXX:5:1214:6786:3014 BX:Z:AAACACCAGACAATAC-1
TCTGTCTCTCTGTCCATCTCTGTCTTTCTATGTCTGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTGTGCCTCTCCCCCCCCCCCCCCCCTCGTCCTTCCCCTCTCCGCCTTCCCCC
+
FAAFAKKKFKKKKFFAAF<AFKF,FFA<FKKAKFKK<AFKFKKKK77F,F7FFFKF<<AF,A,AFAFKKKAFFF,,,,,,,7,7,,7,<<FA,(AAA(,AA(((,,,,,,,,,,(,,,7(((,,,7,7
@E00316:145:HMVG3CCXX:5:1214:6786:3014 BX:Z:AAACACCAGACAATAC-1
AAATGGACAGAGAGAGAAACAGACAGACAGGGAGGGAGGGAGGGGGGCGAGACGGGGGGGGGGAGGGAGGGGGGGAGGGAGGGAGGGGGAGAGACAGAAATAGAGAGAGAGAGAGGGAGAGAGAGAGAGAGAGAAGGGTGGTGGGTGGTGT
+
AAFFFKKKKKKKKKKKKKKKKKKKKKFKKKKKKKKKAFKKF<AF,(,(((<,,,,(((,(,(((,(,,,(,(,,<(,<A,7(,,7(,((,,,,,,7,,7F,,,,,<,7,,A,,,,,,,,<FF<,,,,,<FF<F7,,,,7,,,,,,,,7,7,

This is the format produced by longranger basic.

[shameem356]

Thanks for your help. it is working now.