[habib]

Thank you westerman.

So, each unique kmers would be present in average, at 'coverage depth' many times.

How about several peaks that I observed after depth 1 (which is error kmers)? There are small peak at depth 23, bigger at 46 and highest at 83. Do they indicate that my genome is heterozygous, diploid?

[Brian Bushnell]

Thank you westerman.

So, each unique kmers would be present in average, at 'coverage depth' many times.

How about several peaks that I observed after depth 1 (which is error kmers)? There are small peak at depth 23, bigger at 46 and highest at 83. Do they indicate that my genome is heterozygous, diploid?

It's always difficult to determine exactly what this means. With only a primary peak at 46 and smaller at 23, that indicates a heterozygous diploid. However, the peak at 83 could be a lot of things. Looking at the "unique_kmers column, the first two peaks are actually at 21 and 42, so a 3rd peak at around 84 probably indicates a tetraploid. It could also be a contaminant or an organelle such as mitochondria or chloroplast, but organelles would usually have higher coverage. It could also be 2-copy repeats in the genome. But I suspect it's tetraploid.

[habib]

Thank Brian,

actually there is also another peak at around 1000 coverage, which, as you suggest could be the organelle genomic sequences (I did not include all the data points in my previous post).

With a possibility of tetraploid, I think I am a bit in trouble in how to get a good assembly of this genome...

[DNA Sorcerer]

Hi Brian,

I tried to run bbmap but got an error, and before going into debugging wanted to see if you can tell me if I have a general configuration issue in the custer (e.g. java), so I know what to tell the sysadmin. Thanks a lot in advance.

My line is: CLARKSCV1.2.2-b/bbmap/bbmap.sh ref=contigs.fasta nodisk in1=scratch/s_3_1_sequence.fastq in2=scratch/s_3_2_sequence.fastq covstats=omgen.coverage

And the error message is:
Hello from inside a Grid Engine job running on cl339
Job beginning at Thu Feb 18 16:30:54 NST 2016
Job ending at Thu Feb 18 16:30:54 NST 2016
java -Djava.library.path=/home/cslamovi/CLARKSCV1.2.2-b/bbmap/jni/ -ea -Xmx1310m -cp /home/cslamovi/CLARKSCV1.2.2-b/bbmap/current/ align2.BBMap build=1 overwrite=true fastareadlen=500 ref=contigs.fasta nodisk in1=scratch/s_3_1_sequence.fastq in2=scratch/s_3_2_sequence.fastq covstats=omgen.coverage
Exception in thread "main" java.lang.UnsupportedClassVersionError: Bad version number in .class file
at java.lang.ClassLoader.defineClass1(Native Method)
at java.lang.ClassLoader.defineClass(ClassLoader.java:620)
at java.security.SecureClassLoader.defineClass(SecureClassLoader.java:124)
at java.net.URLClassLoader.defineClass(URLClassLoader.java:260)
at java.net.URLClassLoader.access$100(URLClassLoader.java:56)
at java.net.URLClassLoader$1.run(URLClassLoader.java:195)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:188)
at java.lang.ClassLoader.loadClass(ClassLoader.java:306)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:268)
at java.lang.ClassLoader.loadClass(ClassLoader.java:251)
at java.lang.ClassLoader.loadClassInternal(ClassLoader.java:319)

[GenoMax]

@DNA_sorcerer: @Brian would be along later today with an official answer but first thing to check is what version of java is running on your node/cluster.

Post output of

$  java -version

If I remember this right, @Brian only validates BBMap suite against java v.1.7 and 1.8.

You also may be missing a leading "/" in you file paths (scratch/s_3_1_sequence.fastq) unless "scratch" directory is in the directory from where you are running your command.

[DNA Sorcerer]

Ops, I had forgotten to load java module before. I did now, bbmap run for a couple of minutes and stopped. This is the output:

Hello from inside a Grid Engine job running on cl157
Job beginning at Thu Feb 18 17:03:05 NST 2016
Job ending at Thu Feb 18 17:03:05 NST 2016
java -Djava.library.path=/home/cslamovi/CLARKSCV1.2.2-b/bbmap/jni/ -ea -Xmx1310m -cp /home/cslamovi/CLARKSCV1.2.2-b/bbmap/current/ align2.BBMap build=1 overwrite=true fastareadlen=500 ref=contigs.fasta nodisk in1=scratch/s_3_1_sequence.fastq in2=scratch/s_3_2_sequence.fastq covstats=omgen.coverage
Executing align2.BBMap [build=1, overwrite=true, fastareadlen=500, ref=contigs.fasta, nodisk, in1=scratch/s_3_1_sequence.fastq, in2=scratch/s_3_2_sequence.fastq, covstats=omgen.coverage]

BBMap version 35.82
Retaining first best site only for ambiguous mappings.
No output file.
Executing dna.FastaToChromArrays2 [contigs.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: 0.335 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: 8.592 seconds.
Executing jgi.CoveragePileup [covhist=null, covstats=omgen.coverage, basecov=null, bincov=null, physcov=false, 32bit=false, nzo=false, twocolumn=false, secondary=false, covminscaf=0, ksb=true, binsize=1000, startcov=false, strandedcov=false, rpkm=null, normcov=null, normcovo=null, in1=scratch/s_3_1_sequence.fastq, in2=scratch/s_3_2_sequence.fastq]

Set NONZERO_ONLY to false
Set TWOCOLUMN to false
Set USE_SECONDARY_ALIGNMENTS to false
Set KEEP_SHORT_BINS to true
Set USE_COVERAGE_ARRAYS to false
Set USE_BITSETS to true
Analyzed Index: 6.904 seconds.
Changed from ASCII-33 to ASCII-64 on input quality 97 while prescanning.
Cleared Memory: 1.798 seconds.
Processing reads in paired-ended mode.
Started read stream.
Started 1 mapping thread.

[GenoMax]

Has the grid engine job completed?

Most likely not. After printing the message above BBMap starts loading "genome" index into memory before it starts doing alignments, so be patient .. as long as the job is still "running".

If the job completed then look in the grid engine error file and let us know what is there.

[Brian Bushnell]

Also, note this line:

Started 1 mapping thread.

This means it is running using only 1 thread. Depending on your input size, it could take a while! If that's intentional, then it's fine. But BBMap is fully multithreaded so normally I request all available slots on a node when scheduling, and tell BBMap to use all of them with the flag "threads=16" (for example). It will normally autodetect the number of available processors and use all of them, but on SGE/UGE (which I think you are using), if the NSLOTS environment variable is set, it will cap the max threads at that value (so for example on a 16-core machine, if you request 1 slot, such that "echo $NSLOTS" returns 1, then unless you manually specify the number of threads it will only use 1, to ensure fairness).

I recommend you ssh into the node it's running on and run "top -c", then look for a java process and ensure it is using CPU resources (should be at 100%). You can also examine the output file; it should gradually be growing.

[DNA Sorcerer]

Thanks Brian,
I got it run on 16 threads, but after a little while I get these sort of errors:

Started 16 mapping threads.
Exception in thread "Thread-7" java.lang.AssertionError
at stream.Read.expectedErrors(Read.java:2099)
at stream.Read.avgQualityByProbability(Read.java:1713)
at stream.Read.avgQualityByProbability(Read.java:1706)
at stream.Read.avgQuality(Read.java:1700)
at align2.AbstractMapThread.quickMap(AbstractMapThread.java:688)
at align2.BBMapThread.processReadPair(BBMapThread.java:953)
at align2.AbstractMapThread.run(AbstractMapThread.java:508)
Detecting finished threads: 0Exception in thread "Thread-13" java.lang.AssertionError
at stream.Read.expectedErrors(Read.java:2099)
at stream.Read.avgQualityByProbability(Read.java:1713)
at stream.Read.avgQualityByProbability(Read.java:1706)
at stream.Read.avgQuality(Read.java:1700)
at align2.AbstractMapThread.quickMap(AbstractMapThread.java:688)
at align2.BBMapThread.processReadPair(BBMapThread.java:953)
at align2.AbstractMapThread.run(AbstractMapThread.java:508)
Exception in thread "Thread-12" java.lang.AssertionError
at stream.Read.expectedErrors(Read.java:2099)
at stream.Read.avgQualityByProbability(Read.java:1713)
at stream.Read.avgQualityByProbability(Read.java:1706)
at stream.Read.avgQuality(Read.java:1700)
at align2.AbstractMapThread.quickMap(AbstractMapThread.java:688)
at align2.BBMapThread.processReadPair(BBMapThread.java:953)
at align2.AbstractMapThread.run(AbstractMapThread.java:508)

Before this run the program suggested using ignorebadquality, which I did for this run.

[GenoMax]

Do you know how many cores there are on the node that you are running this on (16 was an example, you may have more or less available on the hardware you have access to)? Are you providing the necessary request for using all available cores on a node in your SGE command line?

Having a multi-threaded job spread out across physical nodes would not be advisable and may lead to the kind of errors you are seeing above.

[DNA Sorcerer]

Having a multi-threaded job spread out across physical nodes would not be advisable and may lead to the kind of errors you are seeing above.

Thanks, I'll take a closer look at the cluster's manual and try again.

[GenoMax]

You don't have to use all of the cores/threads available on a physical server (some clusters may be setup in a way that require you to reserve exclusive access to a node to do that).

I generally find that threads=6 works plenty fast (BBMap is well written). You may run into some I/O limits unless you have a high performance file system that goes along with your HPC cluster.

[Brian Bushnell]

Your error message indicates a problem with the quality scores (admittedly, I should improve the error message from that assertion, but I've never seen it happen before!). Specifically, there are some N bases (no-calls) that have a quality of greater than 0. This could mean that you have an ASCII-64 file being processed as ASCII-33, or it could mean... well, something else, like bug in the base-calling software.

If the problem is just Ns with nonzero quality scores, you can fix the files by running them through Reformat (this command assumes the file is ASCII-33/Sanger):

reformat.sh -da ibq qin=33 in=scratch/s_3_#_sequence.fastq out=fixed_#.fq

Do you happen to know what the quality encoding is of these files? For example, what platform are they from, and how old are they?

[DNA Sorcerer]

Thank Brian,

You are probably right in your suspicion. The read files are old, probably 2011 or so, and were originally .txt and I changed to .fastq. Would this have confused BBmap?

[GenoMax]

Thank Brian,

You are probably right in your suspicion. The read files are old, probably 2011 or so, and were originally .txt and I changed to .fastq. Would this have confused BBmap?

Historically there has been more than one format for Q-scores for fastq files so you need to let BBMap know which score scale to use (most new data is in Sanger fastq format). More on the encoding here: https://en.wikipedia.org/wiki/FASTQ_format#Encoding

@Brian also has a program to identify the correct encoding for your files. That example is in post # 1 of this thread.

$ testformat.sh in=seq.fq.gz