too small output .sai file created by bwa aln? ..leads to bwa sampe hanging?

Stina · 11-29-2012, 03:25 AM

I have sequenced a 6MB region of a single chromosome of the dog genome (targeted capture).

At first I used a fasta file from GenBank of the particular chromosome as a reference sequence to map my reads. I indexed the ref sequence and ran bwa aln, which created a .sai file of about 330 MB for each read. I subsequently ran bwa sampe, which succesfully generated a 9.7 GB .sam file from the paired-end reads.

After this I was recommended to map my reads to the complete genome. So I downloaded the following fasta files from ensemble: each chromosome (1-38), MT, and non-chromosomal DNA and merged the files by cat chr1 chr2 chr3...> mergedFasta.fa. I indexed the fasta file by bwa index and then I ran bwa aln to create .sai files. I got no error messages but noticed that the output file now was 60MB (compared to the 330 .sai file I got when mapping to the single chr GenBank file). I tried to run bwa sampe on the generated .sai files but the program is hanging within 2 minutes. I assume this has to do with the initial creation of the .sai file and the reference FASTA file, but I cant figure out what is wrong? Can somebody help me please?

xied75 · 11-29-2012, 12:24 PM

It might be your merged fa file. Are you running on 32 bit or 64 bit machine? How long does it take to INDEX? Do you still have the output of the index step? How many files generated and what's their sizes? Can you show the *.ann file here?

Stina · 11-30-2012, 12:38 AM

Hi,

Thank you so much for trying to help me.

1) I run Mac OS X 64-bit and bwa is compiled in 64-bit

2) Indexing of my fasta file (bwa index my.fasta) takes about 50 min.

3) Output from indexing:
[bwa_index] Pack FASTA... 38.71 sec
[bwa_index] Construct BWT for the packed sequence...
[BWTIncCreate] textLength=4821953750, availableWord=351290412
[BWTIncConstructFromPacked] 10 iterations done. 99999990 characters processed.
[BWTIncConstructFromPacked] 20 iterations done. 199999990 characters processed.
[BWTIncConstructFromPacked] 30 iterations done. 299999990 characters processed.
[BWTIncConstructFromPacked] 40 iterations done. 399999990 characters processed.
[BWTIncConstructFromPacked] 50 iterations done. 499999990 characters processed.
...
...
[BWTIncConstructFromPacked] 540 iterations done. 4800883702 characters processed.
[BWTIncConstructFromPacked] 550 iterations done. 4821953750 characters processed.
[bwt_gen] Finished constructing BWT in 550 iterations.
[bwa_index] 2105.34 seconds elapse.
[bwa_index] Update BWT... 50.61 sec
[bwa_index] Pack forward-only FASTA... 28.83 sec
[bwa_index] Construct SA from BWT and Occ... 159.11 sec
[main] Version: 0.6.2-r126
[main] CMD: bwa index refGen3.fasta
[main] Real time: 2781.934 sec; CPU: 2382.606 sec

3) generated files from indexing:
refGen3.fasta.amb 393 KB
refGen3.fasta.ann 307 KB
refGen3.fasta.bwt 2.41 GB
refGen3.fasta.pac 602.7 MB
refGen3.fasta.sa 1.21 GB

4) The .ann file is quite long, but here is the start, do you need more?

2410976875 3268 11
0 1 dna:chromosome chromosome:CanFam3.1:1:1:122678785:1 REF
0 122678785 1122
0 2 dna:chromosome chromosome:CanFam3.1:2:1:85426708:1 REF
122678785 85426708 828
0 3 dna:chromosome chromosome:CanFam3.1:3:1:91889043:1 REF
208105493 91889043 707
0 4 dna:chromosome chromosome:CanFam3.1:4:1:88276631:1 REF
299994536 88276631 622
0 5 dna:chromosome chromosome:CanFam3.1:5:1:88915250:1 REF
388271167 88915250 892
0 6 dna:chromosome chromosome:CanFam3.1:6:1:77573801:1 REF
477186417 77573801 786
0 7 dna:chromosome chromosome:CanFam3.1:7:1:80974532:1 REF
554760218 80974532 607
0 8 dna:chromosome chromosome:CanFam3.1:8:1:74330416:1 REF
635734750 74330416 583
0 9 dna:chromosome chromosome:CanFam3.1:9:1:61074082:1 REF
710065166 61074082 764
0 10 dna:chromosome chromosome:CanFam3.1:10:1:69331447:1 REF
771139248 69331447 744
0 11 dna:chromosome chromosome:CanFam3.1:11:1:74389097:1 REF
840470695 74389097 533
0 12 dna:chromosome chromosome:CanFam3.1:12:1:72498081:1 REF
914859792 72498081 482
0 13 dna:chromosome chromosome:CanFam3.1:13:1:63241923:1 REF
987357873 63241923 488
0 14 dna:chromosome chromosome:CanFam3.1:14:1:60966679:1 REF
1050599796 60966679 345
0 15 dna:chromosome chromosome:CanFam3.1:15:1:64190966:1 REF
1111566475 64190966 637
0 16 dna:chromosome chromosome:CanFam3.1:16:1:59632846:1 REF
1175757441 59632846 442
0 17 dna:chromosome chromosome:CanFam3.1:17:1:64289059:1 REF
1235390287 64289059 513
0 18 dna:chromosome chromosome:CanFam3.1:18:1:55844845:1 REF
1299679346 55844845 589
0 19 dna:chromosome chromosome:CanFam3.1:19:1:53741614:1 REF
1355524191 53741614 304
0 20 dna:chromosome chromosome:CanFam3.1:20:1:58134056:1 REF
1409265805 58134056 687
0 21 dna:chromosome chromosome:CanFam3.1:21:1:50858623:1 REF
1467399861 50858623 374
0 22 dna:chromosome chromosome:CanFam3.1:22:1:61439934:1 REF
1518258484 61439934 371
0 23 dna:chromosome chromosome:CanFam3.1:23:1:52294480:1 REF
1579698418 52294480 308
0 24 dna:chromosome chromosome:CanFam3.1:24:1:47698779:1 REF
1631992898 47698779 455
0 25 dna:chromosome chromosome:CanFam3.1:25:1:51628933:1 REF
1679691677 51628933 384
0 26 dna:chromosome chromosome:CanFam3.1:26:1:38964690:1 REF
1731320610 38964690 391
0 27 dna:chromosome chromosome:CanFam3.1:27:1:45876710:1 REF
1770285300 45876710 393
0 28 dna:chromosome chromosome:CanFam3.1:28:1:41182112:1 REF
1816162010 41182112 395
0 29 dna:chromosome chromosome:CanFam3.1:29:1:41845238:1 REF
1857344122 41845238 264
0 30 dna:chromosome chromosome:CanFam3.1:30:1:40214260:1 REF
1899189360 40214260 372
0 31 dna:chromosome chromosome:CanFam3.1:31:1:39895921:1 REF
1939403620 39895921 288
0 32 dna:chromosome chromosome:CanFam3.1:32:1:38810281:1 REF
1979299541 38810281 190
0 33 dna:chromosome chromosome:CanFam3.1:33:1:31377067:1 REF
2018109822 31377067 264
0 34 dna:chromosome chromosome:CanFam3.1:34:1:42124431:1 REF
2049486889 42124431 297
0 35 dna:chromosome chromosome:CanFam3.1:35:1:26524999:1 REF
2091611320 26524999 211
0 36 dna:chromosome chromosome:CanFam3.1:36:1:30810995:1 REF
2118136319 30810995 254
0 37 dna:chromosome chromosome:CanFam3.1:37:1:30902991:1 REF
2148947314 30902991 327
0 38 dna:chromosome chromosome:CanFam3.1:38:1:23914537:1 REF
2179850305 23914537 308
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2203764842 123869142 1032
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0 AAEX03025628.1 dna:scaffold scaffold:CanFam3.1:AAEX03025628.1:1:5446:1 REF
2329633329 5446 0
0 AAEX03025627.1 dna:scaffold scaffold:CanFam3.1:AAEX03025627.1:1:5448:1 REF
2329638775 5448 0
0 AAEX03025623.1 dna:scaffold scaffold:CanFam3.1:AAEX03025623.1:1:5452:1 REF
2329644223 5452 0
0 AAEX03025624.1 dna:scaffold scaffold:CanFam3.1:AAEX03025624.1:1:5452:1 REF
2329649675 5452 0
0 AAEX03025622.1 dna:scaffold scaffold:CanFam3.1:AAEX03025622.1:1:5460:1 REF
2329655127 5460 0
0 AAEX03025621.1 dna:scaffold scaffold:CanFam3.1:AAEX03025621.1:1:5463:1 REF
2329660587 5463 0
0 AAEX03025620.1 dna:scaffold scaffold:CanFam3.1:AAEX03025620.1:1:5476:1 REF
2329666050 5476 0
0 AAEX03025619.1 dna:scaffold scaffold:CanFam3.1:AAEX03025619.1:1:5481:1 REF
2329671526 5481 0
0 AAEX03025618.1 dna:scaffold scaffold:CanFam3.1:AAEX03025618.1:1:5483:1 REF
2329677007 5483 0
0 AAEX03025617.1 dna:scaffold scaffold:CanFam3.1:AAEX03025617.1:1:5485:1 REF
2329682490 5485 0
0 AAEX03025615.1 dna:scaffold scaffold:CanFam3.1:AAEX03025615.1:1:5487:1 REF
2329687975 5487 0
0 AAEX03025616.1 dna:scaffold scaffold:CanFam3.1:AAEX03025616.1:1:5487:1 REF
2329693462 5487 0
0 AAEX03025614.1 dna:scaffold scaffold:CanFam3.1:AAEX03025614.1:1:5489:1 REF
2329698949 5489 0
0 AAEX03025613.1 dna:scaffold scaffold:CanFam3.1:AAEX03025613.1:1:5495:1 REF
2329704438 5495 0
0 AAEX03025612.1 dna:scaffold scaffold:CanFam3.1:AAEX03025612.1:1:5496:1 REF
2329709933 5496 0
0 AAEX03025611.1 dna:scaffold scaffold:CanFam3.1:AAEX03025611.1:1:5497:1 REF
2329715429 5497 0
0 AAEX03025610.1 dna:scaffold scaffold:CanFam3.1:AAEX03025610.1:1:5500:1 REF
2329720926 5500 0
0 AAEX03025609.1 dna:scaffold scaffold:CanFam3.1:AAEX03025609.1:1:5502:1 REF
2329726426 5502 0
0 AAEX03025608.1 dna:scaffold scaffold:CanFam3.1:AAEX03025608.1:1:5517:1 REF
2329731928 5517 0
0 AAEX03025607.1 dna:scaffold scaffold:CanFam3.1:AAEX03025607.1:1:5520:1 REF
2329737445 5520 0
0 AAEX03025606.1 dna:scaffold scaffold:CanFam3.1:AAEX03025606.1:1:5521:1 REF
2329742965 5521 0
0 AAEX03025605.1 dna:scaffold scaffold:CanFam3.1:AAEX03025605.1:1:5522:1 REF
2329748486 5522 0
0 AAEX03025604.1 dna:scaffold scaffold:CanFam3.1:AAEX03025604.1:1:5531:1 REF
2329754008 5531 0
0 AAEX03025602.1 dna:scaffold scaffold:CanFam3.1:AAEX03025602.1:1:5541:1 REF
2329759539 5541 0
0 AAEX03025603.1 dna:scaffold scaffold:CanFam3.1:AAEX03025603.1:1:5541:1 REF
2329765080 5541 0
0 AAEX03025601.1 dna:scaffold scaffold:CanFam3.1:AAEX03025601.1:1:5543:1 REF
2329770621 5543 0
0 AAEX03025600.1 dna:scaffold scaffold:CanFam3.1:AAEX03025600.1:1:5547:1 REF
2329776164 5547 0
0 AAEX03025599.1 dna:scaffold scaffold:CanFam3.1:AAEX03025599.1:1:5548:1 REF
2329781711 5548 0
0 AAEX03025598.1 dna:scaffold scaffold:CanFam3.1:AAEX03025598.1:1:5549:1 REF
2329787259 5549 0
0 AAEX03025596.1 dna:scaffold scaffold:CanFam3.1:AAEX03025596.1:1:5551:1 REF
2329792808 5551 0
0 AAEX03025597.1 dna:scaffold scaffold:CanFam3.1:AAEX03025597.1:1:5551:1 REF
2329798359 5551 0
0 AAEX03025595.1 dna:scaffold scaffold:CanFam3.1:AAEX03025595.1:1:5552:1 REF
2329803910 5552 0
0 AAEX03025594.1 dna:scaffold scaffold:CanFam3.1:AAEX03025594.1:1:5554:1 REF
2329809462 5554 0
0 AAEX03025593.1 dna:scaffold scaffold:CanFam3.1:AAEX03025593.1:1:5556:1 REF
2329815016 5556 0
0 AAEX03025592.1 dna:scaffold scaffold:CanFam3.1:AAEX03025592.1:1:5557:1 REF
2329820572 5557 0
0 AAEX03025591.1 dna:scaffold scaffold:CanFam3.1:AAEX03025591.1:1:5570:1 REF
2329826129 5570 0
0 AAEX03025590.1 dna:scaffold scaffold:CanFam3.1:AAEX03025590.1:1:5571:1 REF
2329831699 5571 0
0 AAEX03025588.1 dna:scaffold scaffold:CanFam3.1:AAEX03025588.1:1:5577:1 REF
2329837270 5577 0
0 AAEX03025589.1 dna:scaffold scaffold:CanFam3.1:AAEX03025589.1:1:5577:1 REF
2329842847 5577 0
0 AAEX03025587.1 dna:scaffold scaffold:CanFam3.1:AAEX03025587.1:1:5578:1 REF
2329848424 5578 0
0 AAEX03025586.1 dna:scaffold scaffold:CanFam3.1:AAEX03025586.1:1:5579:1 REF
2329854002 5579 0
0 AAEX03025585.1 dna:scaffold scaffold:CanFam3.1:AAEX03025585.1:1:5582:1 REF
2329859581 5582 0
0 AAEX03025584.1 dna:scaffold scaffold:CanFam3.1:AAEX03025584.1:1:5584:1 REF
2329865163 5584 0
0 AAEX03025583.1 dna:scaffold scaffold:CanFam3.1:AAEX03025583.1:1:5588:1 REF
2329870747 5588 0
0 AAEX03025582.1 dna:scaffold scaffold:CanFam3.1:AAEX03025582.1:1:5594:1 REF
2329876335 5594 0
0 AAEX03025581.1 dna:scaffold scaffold:CanFam3.1:AAEX03025581.1:1:5605:1 REF
2329881929 5605 0
0 AAEX03025580.1 dna:scaffold scaffold:CanFam3.1:AAEX03025580.1:1:5610:1 REF
2329887534 5610 0
0 AAEX03025578.1 dna:scaffold scaffold:CanFam3.1:AAEX03025578.1:1:5614:1 REF
2329893144 5614 0
0 AAEX03025579.1 dna:scaffold scaffold:CanFam3.1:AAEX03025579.1:1:5614:1 REF
2329898758 5614 0
0 AAEX03025577.1 dna:scaffold scaffold:CanFam3.1:AAEX03025577.1:1:5617:1 REF
2329904372 5617 0
0 AAEX03025576.1 dna:scaffold scaffold:CanFam3.1:AAEX03025576.1:1:5618:1 REF
2329909989 5618 0
0 AAEX03025575.1 dna:scaffold scaffold:CanFam3.1:AAEX03025575.1:1:5622:1 REF
2329915607 5622 0
0 AAEX03025574.1 dna:scaffold scaffold:CanFam3.1:AAEX03025574.1:1:5623:1 REF
2329921229 5623 0
0 AAEX03025573.1 dna:scaffold scaffold:CanFam3.1:AAEX03025573.1:1:5624:1 REF
2329926852 5624 0
0 AAEX03025572.1 dna:scaffold scaffold:CanFam3.1:AAEX03025572.1:1:5627:1 REF
2329932476 5627 0
0 AAEX03025570.1 dna:scaffold scaffold:CanFam3.1:AAEX03025570.1:1:5629:1 REF
2329938103 5629 0
0 AAEX03025571.1 dna:scaffold scaffold:CanFam3.1:AAEX03025571.1:1:5629:1 REF
2329943732 5629 0
0 AAEX03025569.1 dna:scaffold scaffold:CanFam3.1:AAEX03025569.1:1:5630:1 REF
2329949361 5630 0
0 AAEX03025567.1 dna:scaffold scaffold:CanFam3.1:AAEX03025567.1:1:5635:1 REF
2329954991 5635 0
0 AAEX03025568.1 dna:scaffold scaffold:CanFam3.1:AAEX03025568.1:1:5635:1 REF
2329960626 5635 0
0 AAEX03025566.1 dna:scaffold scaffold:CanFam3.1:AAEX03025566.1:1:5638:1 REF
2329966261 5638 0
0 AAEX03025565.1 dna:scaffold scaffold:CanFam3.1:AAEX03025565.1:1:5648:1 REF
2329971899 5648 0
0 AAEX03025564.1 dna:scaffold scaffold:CanFam3.1:AAEX03025564.1:1:5649:1 REF
2329977547 5649 0
0 AAEX03025563.1 dna:scaffold scaffold:CanFam3.1:AAEX03025563.1:1:5650:1 REF
2329983196 5650 0
0 AAEX03025562.1 dna:scaffold scaffold:CanFam3.1:AAEX03025562.1:1:5652:1 REF
2329988846 5652 0
0 AAEX03025561.1 dna:scaffold scaffold:CanFam3.1:AAEX03025561.1:1:5654:1 REF
2329994498 5654 0
0 AAEX03025560.1 dna:scaffold scaffold:CanFam3.1:AAEX03025560.1:1:5656:1 REF
2330000152 5656 0
0 AAEX03025559.1 dna:scaffold scaffold:CanFam3.1:AAEX03025559.1:1:5657:1 REF
2330005808 5657 0
0 AAEX03025558.1 dna:scaffold scaffold:CanFam3.1:AAEX03025558.1:1:5665:1 REF
2330011465 5665 0
0 AAEX03025557.1 dna:scaffold scaffold:CanFam3.1:AAEX03025557.1:1:5667:1 REF
2330017130 5667 0
0 AAEX03025556.1 dna:scaffold scaffold:CanFam3.1:AAEX03025556.1:1:5671:1 REF
2330022797 5671 0
0 AAEX03025555.1 dna:scaffold scaffold:CanFam3.1:AAEX03025555.1:1:5673:1 REF
2330028468 5673 0
0 AAEX03025553.1 dna:scaffold scaffold:CanFam3.1:AAEX03025553.1:1:5674:1 REF
2330034141 5674 0
0 AAEX03025554.1 dna:scaffold scaffold:CanFam3.1:AAEX03025554.1:1:5674:1 REF
2330039815 5674 0
0 AAEX03025552.1 dna:scaffold scaffold:CanFam3.1:AAEX03025552.1:1:5675:1 REF
2330045489 5675 0
0 AAEX03025551.1 dna:scaffold scaffold:CanFam3.1:AAEX03025551.1:1:5677:1 REF
2330051164 5677 0
0 AAEX03025550.1 dna:scaffold scaffold:CanFam3.1:AAEX03025550.1:1:5678:1 REF
2330056841 5678 0
0 AAEX03025549.1 dna:scaffold scaffold:CanFam3.1:AAEX03025549.1:1:5680:1 REF
2330062519 5680 0
0 AAEX03025548.1 dna:scaffold scaffold:CanFam3.1:AAEX03025548.1:1:5681:1 REF
2330068199 5681 0
0 AAEX03025547.1 dna:scaffold scaffold:CanFam3.1:AAEX03025547.1:1:5684:1 REF
2330073880 5684 0
0 AAEX03025544.1 dna:scaffold scaffold:CanFam3.1:AAEX03025544.1:1:5685:1 REF
2330079564 5685 0
0 AAEX03025543.1 dna:scaffold scaffold:CanFam3.1:AAEX03025543.1:1:5688:1 REF
2330085249 5688 0
0 AAEX03025542.1 dna:scaffold scaffold:CanFam3.1:AAEX03025542.1:1:5693:1 REF
2330090937 5693 0
0 AAEX03025541.1 dna:scaffold scaffold:CanFam3.1:AAEX03025541.1:1:5694:1 REF
2330096630 5694 0
0 AAEX03025540.1 dna:scaffold scaffold:CanFam3.1:AAEX03025540.1:1:5695:1 REF
2330102324 5695 0
0 AAEX03025539.1 dna:scaffold scaffold:CanFam3.1:AAEX03025539.1:1:5699:1 REF
2330108019 5699 0
0 JH374193.1 dna:scaffold scaffold:CanFam3.1:JH374193.1:1:5700:1 REF
2330113718 5700 1
0 AAEX03025538.1 dna:scaffold scaffold:CanFam3.1:AAEX03025538.1:1:5703:1 REF
2330119418 5703 0
0 AAEX03025537.1 dna:scaffold scaffold:CanFam3.1:AAEX03025537.1:1:5705:1 REF
2330125121 5705 0
0 AAEX03025536.1 dna:scaffold scaffold:CanFam3.1:AAEX03025536.1:1:5710:1 REF
2330130826 5710 0
0 AAEX03025532.1 dna:scaffold scaffold:CanFam3.1:AAEX03025532.1:1:5711:1 REF
2330136536 5711 0
0 AAEX03025531.1 dna:scaffold scaffold:CanFam3.1:AAEX03025531.1:1:5712:1 REF
2330142247 5712 0
0 AAEX03025530.1 dna:scaffold scaffold:CanFam3.1:AAEX03025530.1:1:5716:1 REF
2330147959 5716 0
0 AAEX03025529.1 dna:scaffold scaffold:CanFam3.1:AAEX03025529.1:1:5721:1 REF
2330153675 5721 0
0 AAEX03025528.1 dna:scaffold scaffold:CanFam3.1:AAEX03025528.1:1:5723:1 REF
2330159396 5723 0
0 AAEX03025527.1 dna:scaffold scaffold:CanFam3.1:AAEX03025527.1:1:5731:1 REF
2330165119 5731 0
0 AAEX03025526.1 dna:scaffold scaffold:CanFam3.1:AAEX03025526.1:1:5733:1 REF
2330170850 5733 0
0 AAEX03025525.1 dna:scaffold scaffold:CanFam3.1:AAEX03025525.1:1:5735:1 REF
2330176583 5735 0
0 AAEX03025524.1 dna:scaffold scaffold:CanFam3.1:AAEX03025524.1:1:5747:1 REF
2330182318 5747 0
0 AAEX03025523.1 dna:scaffold scaffold:CanFam3.1:AAEX03025523.1:1:5748:1 REF
2330188065 5748 0

Do you need more info?
Again, I really appreciate your help!

xied75 · 11-30-2012, 05:00 AM

.ann looks alright seems (at least for the part showing here). Although .ann and .amb sizes are bigger than human genome.

Do you still have the run info of ALN step both for the 330MB and 60MB output?

Stina · 11-30-2012, 09:04 AM

1) Run info (one of the two reads): multifasta file generated from files from ensemble as ref genome sequence (60 MB output .sai file)

bwa aln refGen3.fasta /Volumes/.....-DogCap-1_CGATGT_L003_R1_001.fastq.gz > aln_sa1.sai
[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 262144 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.58 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 524288 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 786432 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 1048576 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 1310720 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 1572864 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 1835008 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 2097152 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 2359296 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.54 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 2621440 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 2883584 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 3145728 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 3407872 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 3670016 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 3932160 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 4194304 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 4456448 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 4718592 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 4980736 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 5242880 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 5505024 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 5767168 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 6029312 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 6291456 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 6553600 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 6815744 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 7077888 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 7340032 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 7602176 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 7864320 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 8126464 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 8388608 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 8650752 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 8912896 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 9175040 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 9437184 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 9699328 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.54 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 9961472 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 10223616 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 10485760 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 10747904 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 11010048 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.58 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 11272192 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 11534336 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 11796480 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 12058624 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 12320768 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 12582912 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 12845056 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 13107200 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 13369344 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 13631488 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 13893632 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 14155776 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.57 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 14417920 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.55 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 14680064 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1.56 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 14942208 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 0.73 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 15064387 sequences have been processed.
[main] Version: 0.6.2-r126
[main] CMD: bwa aln refGen3.fasta /Volumes/.......-DogCap-1_CGATGT_L003_R1_001.fastq.gz
[main] Real time: 188.896 sec; CPU: 156.373 sec

2) Run info (one of the two reads): reference sequence = one fasta file of one chromosome from GenBank (330 MB output .sai file).

bwa aln cf3.fasta /Volumes/...-DogCap-1/Dahlgren-DogCap-1_CGATGT_L003_R1_001.fastq.gz > aln_test1.sai
[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln_core] calculate SA coordinate... 70.50 sec
[bwa_aln_core] write to the disk... 0.04 sec
[bwa_aln_core] 262144 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 70.45 sec
[bwa_aln_core] write to the disk... 0.04 sec
[bwa_aln_core] 524288 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 69.88 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 786432 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 67.47 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 1048576 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 66.83 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 1310720 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 68.28 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 1572864 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 68.40 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 1835008 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 68.76 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 2097152 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 68.45 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 2359296 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 65.06 sec
[bwa_aln_core] write to the disk... 0.04 sec
[bwa_aln_core] 2621440 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.91 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 2883584 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.75 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 3145728 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.32 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 3407872 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.70 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 3670016 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.13 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 3932160 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.35 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 4194304 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 64.04 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 4456448 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.24 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 4718592 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.05 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 4980736 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.17 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 5242880 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.90 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 5505024 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.63 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 5767168 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.43 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 6029312 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.92 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 6291456 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.55 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 6553600 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.43 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 6815744 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 64.90 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 7077888 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.37 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 7340032 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.23 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 7602176 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 60.06 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 7864320 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 60.83 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 8126464 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 64.54 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 8388608 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 66.63 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 8650752 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 66.04 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 8912896 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 66.94 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 9175040 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 65.03 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 9437184 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 64.13 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 9699328 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.01 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 9961472 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.21 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 10223616 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.42 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 10485760 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.12 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 10747904 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.63 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 11010048 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.57 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 11272192 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 64.17 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 11534336 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.45 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 11796480 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.50 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 12058624 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.00 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 12320768 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.32 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 12582912 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.61 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 12845056 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.90 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 13107200 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 60.26 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 13369344 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.79 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 13631488 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 63.02 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 13893632 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.08 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 14155776 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 61.49 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 14417920 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.19 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 14680064 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 62.14 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 14942208 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 30.04 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 15064387 sequences have been processed.
[main] Version: 0.6.2-r126
[main] CMD: bwa aln cf3.fasta /Volumes/...-DogCap-1/Dahlgren-DogCap-1_CGATGT_L003_R1_001.fastq.gz
[main] Real time: 3787.719 sec; CPU: 3740.718 sec

xied75 · 11-30-2012, 09:56 AM

Looks normal again.

Now need to figure out if the .sai file is overwritten or everything inside is garbage. Can you cut 1 million rows from your fastq.gz (you can unzip first), run ALN, see how big the sai is, and then run SAMPE, see what SAM you can get.

swbarnes2 · 11-30-2012, 11:20 AM

Have you tried running samse on the individual .sai files? Maybe it's hanging because the pairing is screwed up?

Stina · 12-01-2012, 01:53 AM

So I cut 1 million rows from my fastq file and ran:

1) bwa aln ...fastq, which generated a 1 MB .sai file (one for each direction).

2) bwa sampe, which generates a 131.6 MB .sam file

bwa sampe refGen3.fasta alnTestFirstOneMilrefGen3_1.sai alnTestFirstOneMilrefGen3_2.sai /...-DogCap-1_CGATGT_L003_R1_001.FirstOneMil.fastq /...-DogCap-1_CGATGT_L003_R2_001.FirstOneMil_R2.fastq > alnFirstOneMil.sam
[bwa_sai2sam_pe_core] convert to sequence coordinate...
[infer_isize] fail to infer insert size: too few good pairs
[bwa_sai2sam_pe_core] time elapses: 2.21 sec
[bwa_sai2sam_pe_core] changing coordinates of 0 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_sai2sam_pe_core] time elapses: 0.63 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 0.87 sec
[bwa_sai2sam_pe_core] print alignments... 0.86 sec
[bwa_sai2sam_pe_core] 250000 sequences have been processed.
[main] Version: 0.6.2-r126 refGen3.fasta alnTestFirstOneMilrefGen3_1.sai alnTestFirstOneMilrefGen3_2.sai /...-DogCap-1_CGATGT_L003_R1_001.FirstOneMil.fastq /...-DogCap-1_CGATGT_L003_R2_001.FirstOneMil_R2.fastq

So why does sampe not work with the entire fastq files?

Swbarnes: bwa sampe works fine when a use a fasta file from GenBank of only one chromosome, but not when I generate a whole genome file of all chromosomes (from ensemble). Do you still think it could be the pairing?

Perhaps I should try samse as well.

Many thanks for the advice i get.

xied75 · 12-01-2012, 05:37 AM

In case you are still working today, 1 million reads to generate 1MB sai file, basically means each read only have a 0x00 there, i.e. not able to align at all, if you do hexdump, you might see lots of 0x00 all over the sai file (after a short header region).

Try: 1, when INDEX, give "-a bwtsw". (EDIT: no need to try this)
2, just cat chr1 + chr2, index, try with the 1million reads again.

Stina · 12-03-2012, 11:37 AM

Hmm.. there is obviously something wrong with my whole-genome fasta file..

1, when INDEX, give "-a bwtsw". (EDIT: no need to try this)
I tried anyway, and as you said it did not work.

2, just cat chr1 + chr2, index, try with the 1million reads again.
I am not sure if this worked?
When I do bwa aln, the output files (.sai) are 5.9 MB (one for each direction). Probably too small?
And when I run bwa sampe, the output .sam file is 161.4 MB.
This is the sampe output:

[bwa_sai2sam_pe_core] convert to sequence coordinate...
[infer_isize] (25, 50, 75) percentile: (258, 290, 331)
[infer_isize] low and high boundaries: 112 and 477 for estimating avg and std
[infer_isize] inferred external isize from 127642 pairs: 293.914 +/- 58.128
[infer_isize] skewness: -0.004; kurtosis: 0.314; ap_prior: 1.36e-05
[infer_isize] inferred maximum insert size: 671 (6.49 sigma)
[bwa_sai2sam_pe_core] time elapses: 3.59 sec
[bwa_sai2sam_pe_core] changing coordinates of 1824 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 15515 out of 18826 Q17 singletons are mated.
[bwa_paired_sw] 1494 out of 2510 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 8.19 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 1.10 sec
[bwa_sai2sam_pe_core] print alignments... 1.67 sec
[bwa_sai2sam_pe_core] 250000 sequences have been processed.
[main] Version: 0.6.2-r126

swbarnes2 · 12-03-2012, 12:00 PM

Quote:

Originally Posted by Stina

Hmm.. there is obviously something wrong with my whole-genome fasta file..

1, when INDEX, give "-a bwtsw". (EDIT: no need to try this)
I tried anyway, and as you said it did not work.

2, just cat chr1 + chr2, index, try with the 1million reads again.
I am not sure if this worked?
When I do bwa aln, the output files (.sai) are 5.9 MB (one for each direction). Probably too small?
And when I run bwa sampe, the output .sam file is 161.4 MB.
This is the sampe output:

[bwa_sai2sam_pe_core] convert to sequence coordinate...
[infer_isize] (25, 50, 75) percentile: (258, 290, 331)
[infer_isize] low and high boundaries: 112 and 477 for estimating avg and std
[infer_isize] inferred external isize from 127642 pairs: 293.914 +/- 58.128
[infer_isize] skewness: -0.004; kurtosis: 0.314; ap_prior: 1.36e-05
[infer_isize] inferred maximum insert size: 671 (6.49 sigma)
[bwa_sai2sam_pe_core] time elapses: 3.59 sec
[bwa_sai2sam_pe_core] changing coordinates of 1824 alignments.
[bwa_sai2sam_pe_core] align unmapped mate...
[bwa_paired_sw] 15515 out of 18826 Q17 singletons are mated.
[bwa_paired_sw] 1494 out of 2510 Q17 discordant pairs are fixed.
[bwa_sai2sam_pe_core] time elapses: 8.19 sec
[bwa_sai2sam_pe_core] refine gapped alignments... 1.10 sec
[bwa_sai2sam_pe_core] print alignments... 1.67 sec
[bwa_sai2sam_pe_core] 250000 sequences have been processed.
[main] Version: 0.6.2-r126

Are you sure you are aligning a million reads? Because the bwa output looks like it is only aligning a quarter million

You realize that a fastq is 4 lines per read, right? So a fastq with a million lines only has a quarter million reads.

xied75 · 12-04-2012, 03:59 AM

Quote:

Are you sure you are aligning a million reads? Because the bwa output looks like it is only aligning a quarter million

You realize that a fastq is 4 lines per read, right? So a fastq with a million lines only has a quarter million reads.

Good catch!

If the library does have an insert size of around 290, then I think the problem is really to do with the whole ref fasta.

Stina · 12-05-2012, 07:32 AM

Thank you so much for trying to help me.

The problem was that I was using bwa version 6.2, which obviously doesnt work properly on a mac, see http://seqanswers.com/forums/showthread.php?t=24133

So I downloaded version 5.10 instead, which solved the problem.

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11-29-2012, 03:25 AM	#1
Stina Junior Member Location: Norway Join Date: Feb 2012 Posts: 9	too small output .sai file created by bwa aln? ..leads to bwa sampe hanging? I have sequenced a 6MB region of a single chromosome of the dog genome (targeted capture). At first I used a fasta file from GenBank of the particular chromosome as a reference sequence to map my reads. I indexed the ref sequence and ran bwa aln, which created a .sai file of about 330 MB for each read. I subsequently ran bwa sampe, which succesfully generated a 9.7 GB .sam file from the paired-end reads. After this I was recommended to map my reads to the complete genome. So I downloaded the following fasta files from ensemble: each chromosome (1-38), MT, and non-chromosomal DNA and merged the files by cat chr1 chr2 chr3...> mergedFasta.fa. I indexed the fasta file by bwa index and then I ran bwa aln to create .sai files. I got no error messages but noticed that the output file now was 60MB (compared to the 330 .sai file I got when mapping to the single chr GenBank file). I tried to run bwa sampe on the generated .sai files but the program is hanging within 2 minutes. I assume this has to do with the initial creation of the .sai file and the reference FASTA file, but I cant figure out what is wrong? Can somebody help me please?

12-01-2012, 05:37 AM	#9
xied75 Senior Member Location: Oxford Join Date: Feb 2012 Posts: 129	In case you are still working today, 1 million reads to generate 1MB sai file, basically means each read only have a 0x00 there, i.e. not able to align at all, if you do hexdump, you might see lots of 0x00 all over the sai file (after a short header region). Try: 1, when INDEX, give "-a bwtsw". (EDIT: no need to try this) 2, just cat chr1 + chr2, index, try with the 1million reads again. Last edited by xied75; 12-03-2012 at 07:53 AM. Reason: to correct mistake.

11-29-2012, 12:24 PM	#2
xied75 Senior Member Location: Oxford Join Date: Feb 2012 Posts: 129	It might be your merged fa file. Are you running on 32 bit or 64 bit machine? How long does it take to INDEX? Do you still have the output of the index step? How many files generated and what's their sizes? Can you show the *.ann file here?

11-30-2012, 05:00 AM	#4
xied75 Senior Member Location: Oxford Join Date: Feb 2012 Posts: 129	.ann looks alright seems (at least for the part showing here). Although .ann and .amb sizes are bigger than human genome. Do you still have the run info of ALN step both for the 330MB and 60MB output?

11-30-2012, 09:04 AM	#5
Stina Junior Member Location: Norway Join Date: Feb 2012 Posts: 9	1) Run info (one of the two reads): multifasta file generated from files from ensemble as ref genome sequence (60 MB output .sai file) bwa aln refGen3.fasta /Volumes/.....-DogCap-1_CGATGT_L003_R1_001.fastq.gz > aln_sa1.sai [bwa_aln] 17bp reads: max_diff = 2 [bwa_aln] 38bp reads: max_diff = 3 [bwa_aln] 64bp reads: max_diff = 4 [bwa_aln] 93bp reads: max_diff = 5 [bwa_aln] 124bp reads: max_diff = 6 [bwa_aln] 157bp reads: max_diff = 7 [bwa_aln] 190bp reads: max_diff = 8 [bwa_aln] 225bp reads: max_diff = 9 [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 262144 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.58 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 524288 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 786432 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 1048576 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 1310720 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 1572864 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 1835008 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 2097152 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 2359296 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.54 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 2621440 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 2883584 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 3145728 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 3407872 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 3670016 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 3932160 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 4194304 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 4456448 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 4718592 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 4980736 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 5242880 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 5505024 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 5767168 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 6029312 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 6291456 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 6553600 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 6815744 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 7077888 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 7340032 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 7602176 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 7864320 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 8126464 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 8388608 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 8650752 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 8912896 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 9175040 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 9437184 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 9699328 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.54 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 9961472 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 10223616 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 10485760 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 10747904 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 11010048 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.58 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 11272192 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 11534336 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 11796480 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 12058624 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 12320768 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 12582912 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 12845056 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 13107200 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 13369344 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 13631488 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 13893632 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 14155776 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.57 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 14417920 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.55 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 14680064 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 1.56 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 14942208 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 0.73 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 15064387 sequences have been processed. [main] Version: 0.6.2-r126 [main] CMD: bwa aln refGen3.fasta /Volumes/.......-DogCap-1_CGATGT_L003_R1_001.fastq.gz [main] Real time: 188.896 sec; CPU: 156.373 sec 2) Run info (one of the two reads): reference sequence = one fasta file of one chromosome from GenBank (330 MB output .sai file). bwa aln cf3.fasta /Volumes/...-DogCap-1/Dahlgren-DogCap-1_CGATGT_L003_R1_001.fastq.gz > aln_test1.sai [bwa_aln] 17bp reads: max_diff = 2 [bwa_aln] 38bp reads: max_diff = 3 [bwa_aln] 64bp reads: max_diff = 4 [bwa_aln] 93bp reads: max_diff = 5 [bwa_aln] 124bp reads: max_diff = 6 [bwa_aln] 157bp reads: max_diff = 7 [bwa_aln] 190bp reads: max_diff = 8 [bwa_aln] 225bp reads: max_diff = 9 [bwa_aln_core] calculate SA coordinate... 70.50 sec [bwa_aln_core] write to the disk... 0.04 sec [bwa_aln_core] 262144 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 70.45 sec [bwa_aln_core] write to the disk... 0.04 sec [bwa_aln_core] 524288 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 69.88 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 786432 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 67.47 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 1048576 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 66.83 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 1310720 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 68.28 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 1572864 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 68.40 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 1835008 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 68.76 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 2097152 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 68.45 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 2359296 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 65.06 sec [bwa_aln_core] write to the disk... 0.04 sec [bwa_aln_core] 2621440 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.91 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 2883584 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.75 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 3145728 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.32 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 3407872 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.70 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 3670016 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.13 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 3932160 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.35 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 4194304 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 64.04 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 4456448 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.24 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 4718592 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.05 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 4980736 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.17 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 5242880 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.90 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 5505024 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.63 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 5767168 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.43 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 6029312 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.92 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 6291456 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.55 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 6553600 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.43 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 6815744 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 64.90 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 7077888 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.37 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 7340032 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.23 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 7602176 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 60.06 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 7864320 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 60.83 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 8126464 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 64.54 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 8388608 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 66.63 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 8650752 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 66.04 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 8912896 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 66.94 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 9175040 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 65.03 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 9437184 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 64.13 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 9699328 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.01 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 9961472 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.21 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 10223616 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.42 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 10485760 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.12 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 10747904 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.63 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 11010048 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.57 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 11272192 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 64.17 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 11534336 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.45 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 11796480 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.50 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 12058624 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.00 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 12320768 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.32 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 12582912 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.61 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 12845056 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.90 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 13107200 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 60.26 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 13369344 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.79 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 13631488 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 63.02 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 13893632 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.08 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 14155776 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 61.49 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 14417920 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.19 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 14680064 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 62.14 sec [bwa_aln_core] write to the disk... 0.03 sec [bwa_aln_core] 14942208 sequences have been processed. [bwa_aln_core] calculate SA coordinate... 30.04 sec [bwa_aln_core] write to the disk... 0.01 sec [bwa_aln_core] 15064387 sequences have been processed. [main] Version: 0.6.2-r126 [main] CMD: bwa aln cf3.fasta /Volumes/...-DogCap-1/Dahlgren-DogCap-1_CGATGT_L003_R1_001.fastq.gz [main] Real time: 3787.719 sec; CPU: 3740.718 sec

11-30-2012, 09:56 AM	#6
xied75 Senior Member Location: Oxford Join Date: Feb 2012 Posts: 129	Looks normal again. Now need to figure out if the .sai file is overwritten or everything inside is garbage. Can you cut 1 million rows from your fastq.gz (you can unzip first), run ALN, see how big the sai is, and then run SAMPE, see what SAM you can get.

11-30-2012, 11:20 AM	#7
swbarnes2 Senior Member Location: San Diego Join Date: May 2008 Posts: 912	Have you tried running samse on the individual .sai files? Maybe it's hanging because the pairing is screwed up?

12-01-2012, 01:53 AM	#8
Stina Junior Member Location: Norway Join Date: Feb 2012 Posts: 9	So I cut 1 million rows from my fastq file and ran: 1) bwa aln ...fastq, which generated a 1 MB .sai file (one for each direction). 2) bwa sampe, which generates a 131.6 MB .sam file bwa sampe refGen3.fasta alnTestFirstOneMilrefGen3_1.sai alnTestFirstOneMilrefGen3_2.sai /...-DogCap-1_CGATGT_L003_R1_001.FirstOneMil.fastq /...-DogCap-1_CGATGT_L003_R2_001.FirstOneMil_R2.fastq > alnFirstOneMil.sam [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] fail to infer insert size: too few good pairs [bwa_sai2sam_pe_core] time elapses: 2.21 sec [bwa_sai2sam_pe_core] changing coordinates of 0 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_sai2sam_pe_core] time elapses: 0.63 sec [bwa_sai2sam_pe_core] refine gapped alignments... 0.87 sec [bwa_sai2sam_pe_core] print alignments... 0.86 sec [bwa_sai2sam_pe_core] 250000 sequences have been processed. [main] Version: 0.6.2-r126 refGen3.fasta alnTestFirstOneMilrefGen3_1.sai alnTestFirstOneMilrefGen3_2.sai /...-DogCap-1_CGATGT_L003_R1_001.FirstOneMil.fastq /...-DogCap-1_CGATGT_L003_R2_001.FirstOneMil_R2.fastq So why does sampe not work with the entire fastq files? Swbarnes: bwa sampe works fine when a use a fasta file from GenBank of only one chromosome, but not when I generate a whole genome file of all chromosomes (from ensemble). Do you still think it could be the pairing? Perhaps I should try samse as well. Many thanks for the advice i get.

12-03-2012, 11:37 AM	#10
Stina Junior Member Location: Norway Join Date: Feb 2012 Posts: 9	Hmm.. there is obviously something wrong with my whole-genome fasta file.. 1, when INDEX, give "-a bwtsw". (EDIT: no need to try this) I tried anyway, and as you said it did not work. 2, just cat chr1 + chr2, index, try with the 1million reads again. I am not sure if this worked? When I do bwa aln, the output files (.sai) are 5.9 MB (one for each direction). Probably too small? And when I run bwa sampe, the output .sam file is 161.4 MB. This is the sampe output: [bwa_sai2sam_pe_core] convert to sequence coordinate... [infer_isize] (25, 50, 75) percentile: (258, 290, 331) [infer_isize] low and high boundaries: 112 and 477 for estimating avg and std [infer_isize] inferred external isize from 127642 pairs: 293.914 +/- 58.128 [infer_isize] skewness: -0.004; kurtosis: 0.314; ap_prior: 1.36e-05 [infer_isize] inferred maximum insert size: 671 (6.49 sigma) [bwa_sai2sam_pe_core] time elapses: 3.59 sec [bwa_sai2sam_pe_core] changing coordinates of 1824 alignments. [bwa_sai2sam_pe_core] align unmapped mate... [bwa_paired_sw] 15515 out of 18826 Q17 singletons are mated. [bwa_paired_sw] 1494 out of 2510 Q17 discordant pairs are fixed. [bwa_sai2sam_pe_core] time elapses: 8.19 sec [bwa_sai2sam_pe_core] refine gapped alignments... 1.10 sec [bwa_sai2sam_pe_core] print alignments... 1.67 sec [bwa_sai2sam_pe_core] 250000 sequences have been processed. [main] Version: 0.6.2-r126

12-05-2012, 07:32 AM	#13
Stina Junior Member Location: Norway Join Date: Feb 2012 Posts: 9	Thank you so much for trying to help me. The problem was that I was using bwa version 6.2, which obviously doesnt work properly on a mac, see http://seqanswers.com/forums/showthread.php?t=24133 So I downloaded version 5.10 instead, which solved the problem.