[Brian Bushnell]

Hi Kate,

That's odd... I will investigate and see if I can figure out why that's happening. minid and idfilter are slightly different, but certainly with idfilter=0.95 you should not get any alignments with under 95% identity.

-Brian

[sk8bro]

Thanks so much! I can try to replicate the issue and email you a small dataset/reference if that would help? Just let me know. BBsplit is otherwise really fantastic!

Kate

[Brian Bushnell]

Hi Kate,

Yes, please do that - it would be very helpful.

-Brian

[schalivendra]

BBMap

Hi,

How I can cite BBMap? Is there a publication that I can list under references?

thanks very much,
SC

[Brian Bushnell]

Hi SC,

No publication yet, so for now, please cite it like this:

"BBMap - Bushnell B. - sourceforge.net/projects/bbmap/"

...or similar, depending on the style guidelines.

[nlubock]

BBMap Error

Hi Brian,

I'm getting the following error when using BBMap (Version 36.11) for some of my alignments:

java -Djava.library.path=/usr/local/stow/bbmap_36.11/bin/jni/ -ea -Xmx8g -cp /usr/local/stow/bbmap_36.11/bin/current/ align2.BBMap build=1 overwrite=true fastareadlen=500 in=1_nonDoped.adapters.fastq vslow=t k=8 ref=../ref-seq.fasta mdtag=t -Xmx8g outm=bbmap.sam nodisk
Executing align2.BBMap [tipsearch=150, minhits=1, minratio=0.25, rescuemismatches=50, rescuedist=3000, build=1, overwrite=true, fastareadlen=500, in=1_nonDoped.adapters.fastq, k=8, ref=../ref-seq.fasta, mdtag=t, -Xmx8g, outm=bbmap.sam, nodisk]

BBMap version 36.11
Set MINIMUM_ALIGNMENT_SCORE_RATIO to 0.250
Retaining first best site only for ambiguous mappings.
Executing dna.FastaToChromArrays2 [../ref-seq.fasta, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=false, minscaf=1, midpad=300, startpad=8000, stoppad=8000, nodisk=true]

Set genScaffoldInfo=true
Set genome to 1

Loaded Reference:	0.001 seconds.
Loading index for chunk 1-1, build 1
Indexing threads started for block 0-1
Indexing threads finished for block 0-1
Generated Index:	0.013 seconds.
Analyzed Index:   	0.031 seconds.
Started output stream:	0.022 seconds.
Cleared Memory:    	0.123 seconds.
Processing reads in single-ended mode.
Started read stream.
Started 40 mapping threads.
Exception in thread "Thread-25" java.lang.ArrayIndexOutOfBoundsException: 136
	at align2.BBIndex.getOffsetArray(BBIndex.java:3065)
	at align2.BBIndex.shrink2(BBIndex.java:830)
	at align2.BBIndex.find(BBIndex.java:443)
	at align2.BBIndex.findAdvanced(BBIndex.java:396)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:736)
	at align2.BBMapThread.processRead(BBMapThread.java:405)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Detecting finished threads: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39

**************************************************************************
Warning!  1 mapping thread did not terminate normally.
Check the error log; the output may be corrupt or incomplete.
Please submit the full stderr output as a bug report, not just this message.
**************************************************************************




   ------------------   Results   ------------------

Genome:                	1
Key Length:            	8
Max Indel:             	16000
Minimum Score Ratio:  	0.25
Mapping Mode:         	normal
Reads Used:           	570587	(170899784 bases)

Mapping:          	34.755 seconds.
Reads/sec:       	16417.62
kBases/sec:      	4917.33


Read 1 data:      	pct reads	num reads 	pct bases	   num bases

mapped:          	 99.9855% 	   570504 	 99.9840% 	   170872487
unambiguous:     	 99.9855% 	   570504 	 99.9840% 	   170872487
ambiguous:       	  0.0000% 	        0 	  0.0000% 	           0
low-Q discards:  	  0.0000% 	        0 	  0.0000% 	           0

perfect best site:	 62.1351% 	   354535 	 62.2356% 	   106360500
semiperfect site:	 62.1351% 	   354535 	 62.2356% 	   106360500

Match Rate:      	      NA 	       NA 	 99.7089% 	   170661054
Error Rate:      	 37.4881% 	   213882 	  0.2887% 	      494106
Sub Rate:        	 28.9387% 	   165105 	  0.1164% 	      199217
Del Rate:        	 10.8304% 	    61791 	  0.1676% 	      286801
Ins Rate:        	  1.1807% 	     6736 	  0.0047% 	        8088
N Rate:          	  0.6976% 	     3980 	  0.0024% 	        4128
Exception in thread "main" java.lang.AssertionError:
The number of reads out does not add up to the number of reads in.
This may indicate that a mapping thread crashed.
If you submit a bug report, include the entire console output, not just this error message.
570504+0+0+82+0 = 570586 != 570587
	at align2.AbstractMapper.printOutput(AbstractMapper.java:1867)
	at align2.BBMap.testSpeed(BBMap.java:489)
	at align2.BBMap.main(BBMap.java:33)

Any insights would be greatly appreciated!

[Brian Bushnell]

It looks like the problem is that some of the internal buffers were not sized correctly for a situation with such long reads and such a short kmer length in conjunction with vslow (which increases the number of seed kmers used per read). You can probably resolve this by using mapPacBio.sh instead of bbmap.sh, which uses larger buffers. How long are the sequences you are mapping? It looks like almost all of them are ~300bp long, but perhaps one read is longer? Also... what are you mapping, and to what?

Alternatively, using a longer kmer length (such as the default of 13) seems like it would be fine in this case. 62% of your reads match the reference perfectly and there's an overall average error rate of 0.288% (average identity of 99.71%) so it does not seem like such extremely sensitive settings are necessary, though it depends on what you are doing. Decreasing K can improve sensitivity when dealing with extremely high error rates, but strange things (like this buffer overflow) can happen if you make it too low. bbmap.sh is tuned for normal Illumina reads being mapped to a genome or transcriptome. For situations with extremely high error rates, or when very small kmer lengths are needed, or with very long reads, I recommend using mapPacBio.sh instead. Also, for small values of K (below 11) I recommend reducing "maxindel" as well.

That said, I'll make this buffer slightly bigger - hopefully that will circumvent this problem in the future.

[nlubock]

Thanks for the quick response!

I'm basically trying to detect every error in a single amplicon with Illumina sequencing. Ideally, I would be able to map every read back to the reference and use the resulting alignments to quantify the errors. I tried to max out every sensitivity parameter in hopes of catching as many errors as possible.

In this case, our reads should be ~300 bp and I'm mapping them onto a single perfect 300 bp reference. Upping the kmer size to 9 with bbmap.sh avoids the overflow, although mapPacBio.sh is throwing this error:

java -Djava.library.path=/usr/local/stow/bbmap_36.11/bin/jni/ -ea -Xmx79769m -cp /usr/local/stow/bbmap_36.11/bin/current/ align2.BBMapPacBio build=1 overwrite=true minratio=0.40 fastareadlen=6000 ambiguous=best minscaf=100 startpad=10000 stoppad=10000 midpad=6000 in=../2_merge/1_nonDoped.adapters.fastq ref=../ref-seq.fasta vslow=t t=30 k=8 outm=pacbio.sam overwrite=t mdtag=t maxindel=100 nodisk
Executing align2.BBMapPacBio [tipsearch=22, minhits=1, minratio=0.25, rescuemismatches=50, rescuedist=3000, build=1, overwrite=true, minratio=0.40, fastareadlen=6000, ambiguous=best, minscaf=100, startpad=10000, stoppad=10000, midpad=6000, in=../2_merge/1_nonDoped.adapters.fastq, ref=../ref-seq.fasta, t=30, k=8, outm=pacbio.sam, overwrite=t, mdtag=t, maxindel=100, nodisk]

BBMap version 36.11
Set MINIMUM_ALIGNMENT_SCORE_RATIO to 0.250
Set MINIMUM_ALIGNMENT_SCORE_RATIO to 0.400
Set threads to 30
Retaining first best site only for ambiguous mappings.
Executing dna.FastaToChromArrays2 [../ref-seq.fasta, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=false, minscaf=100, midpad=6000, startpad=10000, stoppad=10000, nodisk=true]

Set genScaffoldInfo=true
Set genome to 1

Loaded Reference:	0.001 seconds.
Loading index for chunk 1-1, build 1
Indexing threads started for block 0-1
Indexing threads finished for block 0-1
Generated Index:	0.011 seconds.
Analyzed Index:   	0.024 seconds.
Started output stream:	0.014 seconds.
Cleared Memory:    	0.132 seconds.
Processing reads in single-ended mode.
Started read stream.
Started 30 mapping threads.
Exception in thread "Thread-6" Exception in thread "Thread-35" Exception in thread "Thread-34" Exception in thread "Thread-33" Exception in thread "Thread-32" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-31" Detecting finished threads: 0java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-30" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-29" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-28" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-27" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-26" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-25" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-24" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-23" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-22" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-21" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-19" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-20" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-18" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-17" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-15" Exception in thread "Thread-16" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-14" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-13" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-12" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-11" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-10" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-9" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-8" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
Exception in thread "Thread-7" java.lang.AssertionError: 10.75, 8
	at align2.KeyRing.desiredKeysFromDensity(KeyRing.java:271)
	at align2.KeyRing.makeOffsets3(KeyRing.java:437)
	at align2.AbstractMapThread.quickMap(AbstractMapThread.java:683)
	at align2.BBMapThreadPacBio.processRead(BBMapThreadPacBio.java:399)
	at align2.AbstractMapThread.run(AbstractMapThread.java:495)
, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29

**************************************************************************
Warning!  30 mapping threads did not terminate normally.
Check the error log; the output may be corrupt or incomplete.
Please submit the full stderr output as a bug report, not just this message.
**************************************************************************




   ------------------   Results   ------------------

Genome:                	1
Key Length:            	8
Max Indel:             	100
Minimum Score Ratio:  	0.4
Mapping Mode:         	normal
Reads Used:           	30	(8996 bases)

Mapping:          	17.908 seconds.
Reads/sec:       	1.68
kBases/sec:      	0.50


Read 1 data:      	pct reads	num reads 	pct bases	   num bases

mapped:          	  0.0000% 	        0 	  0.0000% 	           0
unambiguous:     	  0.0000% 	        0 	  0.0000% 	           0
ambiguous:       	  0.0000% 	        0 	  0.0000% 	           0
low-Q discards:  	  0.0000% 	        0 	  0.0000% 	           0

perfect best site:	  0.0000% 	        0 	  0.0000% 	           0
semiperfect site:	  0.0000% 	        0 	  0.0000% 	           0

Match Rate:      	      NA 	       NA 	     NaN% 	           0
Error Rate:      	     NaN% 	        0 	     NaN% 	           0
Sub Rate:        	     NaN% 	        0 	     NaN% 	           0
Del Rate:        	     NaN% 	        0 	     NaN% 	           0
Ins Rate:        	     NaN% 	        0 	     NaN% 	           0
N Rate:          	     NaN% 	        0 	     NaN% 	           0
Exception in thread "main" java.lang.AssertionError:
The number of reads out does not add up to the number of reads in.
This may indicate that a mapping thread crashed.
If you submit a bug report, include the entire console output, not just this error message.
0+0+0+0+0 = 0 != 30
	at align2.AbstractMapper.printOutput(AbstractMapper.java:1867)
	at align2.BBMapPacBio.testSpeed(BBMapPacBio.java:458)
	at align2.BBMapPacBio.main(BBMapPacBio.java:35)

[susanklein]

miRNA

Hi,

has anyone used bbmap to map miRNAs to mirDB or similar. Any idea what settings would be best? I need to get count data.

Thanks,

S.

[GenoMax]

Hi,

has anyone used bbmap to map miRNAs to mirDB or similar. Any idea what settings would be best? I need to get count data.

Thanks,

S.

@Brian recently offered this advice on this topic. You may want to modify the maxsites to suite your requirement.

When mapping small RNA's with BBMap use the following flags to report only perfect matches.

Code:
ambig=all vslow perfectmode maxsites=1000

Lot of these gems are captured in this thread.

[susanklein]

thanks, good to know bbmap should work, it is my favourite mapper. I'll try those parameters.

S.

[Brian Bushnell]

Thanks, nlubock; you've allowed me to resolve 2 unexpected corner-cases with very short kmer lengths. I've released a new version (36.14) fixing both of them.

[nlubock]

Thank you! Love the product.

I'm not sure if this is a bug as well, but I noticed that some of the MD Tags in my output files are missing 0's between adjacent mismatches. However, if there is a deletion and then a mismatch, bbmap.sh will add a 0.

For example, if I align the following fasta file:

>perf
CATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAAGCCT
>mm-at-25-26
CATCAGGACCAAGACTGGGGAGATGGTCTCTGCTGTCCACATTGAAGCCT
>del-24-mm-at-25-26
CATCAGGACCAAGACTGGGGAGAGGTCTCTGCTGTCCACATTGAAGCCT
>del-24-mm-at-25
CATCAGGACCAAGACTGGGGAGAGATCTCTGCTGTCCACATTGAAGCCT

back onto the perfect sequence, and then run the resulting sam file through samtools calmd, I get:

50
24CA24    -> 24C0A24
23^T0CA24 -> 23^T0C0A24
23^T0C25

Maybe this is a samtools convention? The official spec was not very clear...

[Brian Bushnell]

There is no official spec for MD tags as far as I know; it's basically defined by usage. The sam PDF just provides vague guidance. I think 0's are optional for adjacent mismatches and there is no indication otherwise in the sam spec. For a deletion followed by a mismatch, the 0 allows you to visually determine how long the deletion is without looking at the cigar string (otherwise it would be hard to visually distinguish between ^AC meaning "deletion of A followed by mismatch of C" versus "deletion of AC". But when combined with the cigar string there is no ambiguity, so they are both correct. Especially considering MD tags have no formal specification For machine-processing, there's no reason to have 0's and they just waste space.

[parulagwl]

We have been liking our first experiences with BBmap for our wheat data and are impressed. And it works well when we use parts of a wheat chromosome for both indexing and mapping. However, when we move from using parts of a chromosome to a whole chromosome (e.g. chr 3B approx. 850 million bases) we get the following error during indexing:

bbmap.sh k=14 ref=3B.fa

java -Djava.library.path=/software/bbmap/bbmap/jni/ -ea -Xmx119912m -cp /software/bbmap/bbmap/current/ align2.BBMap build=1 overwrite=true fastareadlen=500 k=14 ref=3B.fa

Executing align2.BBMap [build=1, overwrite=true, fastareadlen=500, k=14, ref=3B.fa]

BBMap version 35.74

Retaining first best site only for ambiguous mappings.
No output file.

NOTE: Ignoring reference file because it already appears to have been processed.
NOTE: If you wish to regenerate the index, please manually delete ref/genome/1/summary.txt

Set genome to 1

Loaded Reference: 0.008 seconds.

Exception in thread "main" java.lang.AssertionError: 1, 0
at align2.BBIndex.loadIndex(BBIndex.java:94)
at align2.BBMap.loadIndex(BBMap.java:361)
at align2.BBMap.main(BBMap.java:31)

If we split Chr 3B into two fasta sequences and run it works fine and dandy. Have also tried BBmap v36.20 and get the same error. I'm guessing there is a limitation on Chr size? Is there a way to adjust this?
Many Thanks.