Bfast

[jsun529]

Can any one share a quick guide on how to install and run BFAST for SOLiD data? The book does not give a clear instruction so does the readme file. If we do target resequencing, not the complete genome rather genes how should the reference file be prepared. Thanks much in advance.

[nilshomer]

Can any one share a quick guide on how to install and run BFAST for SOLiD data? The book does not give a clear instruction so does the readme file. If we do target resequencing, not the complete genome rather genes how should the reference file be prepared. Thanks much in advance.

If you find something unclear, please let us know so we can improve the manual (email: [email protected]). For targetting resequencing, you must consider whether to use the full reference, or only the targetted region. The former is more computationally expensive, while the latter may have biases if your targetting was off.

The easiest way to run BFAST in a "targetted" mode is to create indexes that only use the targetted regions. This can be accomplished using the "-x" option in when running "bfast index". Example input files can be found in the manual.

[jsun529]

So, the bfast-book or the readme did not say how to install the program, also for the ABI solid example section 7.1.2, for the bpreprocess or bmatches, could not find any of this command from the source code downloaded through sourceforge for the latest version?.

[jsun529]

on the INSTALL page it said refer to the man pages for documentation

doc/bpreprocess.1

but where is this doc folder, this is really confusing and not user friendly and it would be better everything are in one place, say the bfast book or something.

Thanks,

[jsun529]

I have the preprocess running however i got this error message when I trying to create the indexes, any one know how to fix this? Thanks.(I have fragment data not mate pair)

bfast-0.5.6/bpreprocess/bpreprocess -r bfast.rg.file.OPA1.1.brg -i layouts.txt -a 1 -A 1 -o OPA1 -d ./ -T ./
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.1.brg.
Validating indexLayoutFileName layouts.txt.
Validating exonsFileName Default.txt.
Validating outputID OPA1.
Validating outputDir ./.
Validating tmpDir path ./.
Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.1.brg
algorithm: 1
space: 1
indexLayoutFileName: layouts.txt
repeatMasker: 0
startContig: 0
startPos: 0
endContig: 2147483647
endPos: 2147483647
exonsFileName: Default.txt
numThreads: 1
outputID: OPA1
outputDir: ./
tmpDir: ./
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.1.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
************************************************************
In function "RGIndexLayoutRead": Fatal Error[OpenFileError]. Message: Could not open index layout file reading.
***** Exiting due to errors *****
************************************************************

[nilshomer]

I have the preprocess running however i got this error message when I trying to create the indexes, any one know how to fix this? Thanks.(I have fragment data not mate pair)

bfast-0.5.6/bpreprocess/bpreprocess -r bfast.rg.file.OPA1.1.brg -i layouts.txt -a 1 -A 1 -o OPA1 -d ./ -T ./
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.1.brg.
Validating indexLayoutFileName layouts.txt.
Validating exonsFileName Default.txt.
Validating outputID OPA1.
Validating outputDir ./.
Validating tmpDir path ./.
Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.1.brg
algorithm: 1
space: 1
indexLayoutFileName: layouts.txt
repeatMasker: 0
startContig: 0
startPos: 0
endContig: 2147483647
endPos: 2147483647
exonsFileName: Default.txt
numThreads: 1
outputID: OPA1
outputDir: ./
tmpDir: ./
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.1.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
************************************************************
In function "RGIndexLayoutRead": Fatal Error[OpenFileError]. Message: Could not open index layout file reading.
***** Exiting due to errors *****
************************************************************

It looks like your layout file is not properly formatted (there are examples in the manual!). Please post the layout file when you get the chance.

I would suggests upgrading to the 0.6.0 version as the user interface has been improved.

[jsun529]

Thanks, what would be a good index layout for 50bp SOLiD reads?

[nilshomer]

Recommended settings can be found in the manual.

[jsun529]

Thanks. If I were to look for a 20 ~60bp deletion or insertion, how would this reflect to the score matrix for the gap penalty,etc. I assume the example is generic, did not specify.

Best,

[jsun529]

Also what would be a good/valid offset for 50bp solid data the example goes with
1 3 5 7 9 11 13 15 17 19
Will this be good enough?

[jsun529]

Ok, when running the bmatches I got this error message using the example settings.

sudo bfast-0.5.6/bmatches/bmatches -r bfast.rg.file.OPA1.1.brg -i main.indexes.txt -I secondary.indexes.txt -R OPA1F3.1.fastq -O offsets.txt -A 1 -K 8 -M 384 -o OPA1.1 -d ./ -T ./
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.1.brg.
Validating bfastMainIndexesFileName main.indexes.txt.
Validating bfastSecondaryIndexesFileName secondary.indexes.txt.
Validating readsFileName OPA1F3.1.fastq.
Validating offsetsFileName offsets.txt.
Validating outputID OPA1.1.
Validating outputDir ./.
Validating tmpDir path ./.
**** Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.1.brg
bfastMainIndexesFileName main.indexes.txt
bfastSecondaryIndexesFileName secondary.indexes.txt
readsFileName: OPA1F3.1.fastq
offsetsFileName: offsets.txt
space: 1
startReadNum: -1
endReadNum: -1
keySize: 0
maxKeyMatches: 8
maxNumMatches: 384
whichStrand: 0 [BothStrands]
numThreads: 1
queueLength: 100000
outputID: OPA1.1
outputDir: ./
tmpDir: ./
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.1.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
Reading OPA1F3.1.fastq into temp files.
Will process 1933855 reads.
************************************************************
Will output to ./bfast.matches.file.OPA1.1.bmf.
************************************************************
Processing 1933855 reads using 20 main indexes.
************************************************************
Searching index 1 out of 20...
Reading index from 14.
************************************************************
In function "RGIndexRead": Fatal Error[OpenFileError]. Variable/Value: 14.
Message: Could not open rgIndexFileName for reading.
***** Exiting due to errors *****
************************************************************

any suggestions? Thanks,

[nilshomer]

Ok, when running the bmatches I got this error message using the example settings.

sudo bfast-0.5.6/bmatches/bmatches -r bfast.rg.file.OPA1.1.brg -i main.indexes.txt -I secondary.indexes.txt -R OPA1F3.1.fastq -O offsets.txt -A 1 -K 8 -M 384 -o OPA1.1 -d ./ -T ./
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.1.brg.
Validating bfastMainIndexesFileName main.indexes.txt.
Validating bfastSecondaryIndexesFileName secondary.indexes.txt.
Validating readsFileName OPA1F3.1.fastq.
Validating offsetsFileName offsets.txt.
Validating outputID OPA1.1.
Validating outputDir ./.
Validating tmpDir path ./.
**** Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.1.brg
bfastMainIndexesFileName main.indexes.txt
bfastSecondaryIndexesFileName secondary.indexes.txt
readsFileName: OPA1F3.1.fastq
offsetsFileName: offsets.txt
space: 1
startReadNum: -1
endReadNum: -1
keySize: 0
maxKeyMatches: 8
maxNumMatches: 384
whichStrand: 0 [BothStrands]
numThreads: 1
queueLength: 100000
outputID: OPA1.1
outputDir: ./
tmpDir: ./
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.1.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
Reading OPA1F3.1.fastq into temp files.
Will process 1933855 reads.
************************************************************
Will output to ./bfast.matches.file.OPA1.1.bmf.
************************************************************
Processing 1933855 reads using 20 main indexes.
************************************************************
Searching index 1 out of 20...
Reading index from 14.
************************************************************
In function "RGIndexRead": Fatal Error[OpenFileError]. Variable/Value: 14.
Message: Could not open rgIndexFileName for reading.
***** Exiting due to errors *****
************************************************************

any suggestions? Thanks,

Again, please upgrade to bfast-0.6.0d

1. I would recommend using all possible offsets.

2. Long gaps 60bp will be difficult to detect with short reads directly.

3. It looks like the "main.indexes.txt" file is incorrectly formatted. It should just be file paths.

[jsun529]

Thanks. This will be my last question for the test run I think :-)
when I do this bpostprocess as recommended by the book I got these error message as

ocalhost:~/Desktop/OPA1.BFAST # bfast-0.5.6/bpostprocess/bpostprocess -i bfast.aligned.file.OPA1.baf -r bfast.rg.file.OPA1.0.brg -a 3 -o OPA1 -d ./ -O 3
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.0.brg.
Validating alignFileName bfast.aligned.file.OPA1.baf.
Validating outputID OPA1.
Validating outputDir path ./.
Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.0.brg
alignFileName: bfast.aligned.file.OPA1.baf
algorithm: 3 [Best Score]
queueLength: 10000
outputID: OPA1
outputDir: ./
outputFormat: 6
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.0.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
Processing reads, currently on:
0************************************************************
In function "ConvertReadFromColorSpace": Fatal Error[OutOfRange]. Variable/Value: read.
Message: Could not convert base and color.
***** Exiting due to errors *****
************************************************************
not sure which part cause this? Thanks

[nilshomer]

Thanks. This will be my last question for the test run I think :-)
when I do this bpostprocess as recommended by the book I got these error message as

ocalhost:~/Desktop/OPA1.BFAST # bfast-0.5.6/bpostprocess/bpostprocess -i bfast.aligned.file.OPA1.baf -r bfast.rg.file.OPA1.0.brg -a 3 -o OPA1 -d ./ -O 3
************************************************************
Checking input parameters supplied by the user ...
Validating rgFileName bfast.rg.file.OPA1.0.brg.
Validating alignFileName bfast.aligned.file.OPA1.baf.
Validating outputID OPA1.
Validating outputDir path ./.
Input arguments look good!
************************************************************
************************************************************
Printing Program Parameters:
programMode: 1 [ExecuteProgram]
rgFileName: bfast.rg.file.OPA1.0.brg
alignFileName: bfast.aligned.file.OPA1.baf
algorithm: 3 [Best Score]
queueLength: 10000
outputID: OPA1
outputDir: ./
outputFormat: 6
timing: 0
************************************************************
************************************************************
Reading in reference genome from bfast.rg.file.OPA1.0.brg.
In total read 1 contigs for a total of 104204 bases
************************************************************
Processing reads, currently on:
0************************************************************
In function "ConvertReadFromColorSpace": Fatal Error[OutOfRange]. Variable/Value: read.
Message: Could not convert base and color.
***** Exiting due to errors *****
************************************************************
not sure which part cause this? Thanks

This seems to be a bug in bfast version 0.5.6. I am really trying to get you to upgrade to the latest version (0.6.0d) as most of these problems and pitfalls are gone!

[jsun529]

when i use samtools.pl varFilter to filter the variant and snps, it seemed the program have a cap on the maximum read depth, it filtered most of the candidates that has higher coverage that should not be filtered, however when I try to redefine the D through command line arg, the program return nothing after filtering, any one know how to fix it?

Thanks