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=== Purpose ===
= Overview of the <code>bam2FastQ</code> function of <code>[[bamUtil]]</code> =
The <code>bam2FastQ</code> option on the [[bamUtil]] converts a BAM file into FastQ files. This is necessary when only BAM files are delivered but a new alignment is desired. By converting BAM to FastQ files new alignments can be done using FastQ files
'''NOTE: Secondary and Supplementary reads are skipped when converting to FastQ. It assumes that there will only be 2 reads (the 2 primary mates) with the same read name that are not secondary or supplementary.'''
'''NOTE: Use the --splitRG option to split reads into read group specific FASTQs.'''
== How to use it ==
== How to use it ==
When bam2FastQ is invoked without any arguments the following information is displayed
When bam2FastQ is invoked without any arguments the usage information is displayed as described below under [[#Usage|Usage]].
The input BAM file is required, [[#input File (--in)|input File (--in)]].
It works on both read/query name and coordinate sorted SAM/BAM files.
If you want to convert a SAM/BAM that is read/query name sorted but the SO field of the header does not specify "queryname", then use the [[#BAM File Is Sorted By Read Name (--readname)|--readName]] option.
When processing files sorted by read name, the only requirement is that matching read names are next to each other. It does not need to be in strict alphabetical order.
Read Names in paired-end FASTQ files are appended with "/1" for the first in the pair, and "/2" for the second in the pair. Override these defaults using [[#First in Pair FastQ ReadName Extension (--firstRNExt)|--firstRNExt]] and [[#Second in Pair FastQ ReadName Extension (--secondRNExt)|--secondRNExt]]
Sequences marked as Reverse strands in the SAM/BAM file are reverse complemented prior to writing to the FASTQ files. To skip this step, specify [[#Do Not Reverse Complement Reverse Strands (--noReverseComp)|--noReverseComp]]
Any errors and a summary of how many pairs and unpaired reads were processed are written to stderr.
'''NOTE: This tool does not work on templates that have more than 2 segments. It does not properly match reads when more than 2 reads have the same read name.'''
'''NOTE: This tool does not split reads into read group specific FASTQs. If you want Read Group specific FASTQ files, first run [[BamUtil: splitBam]] to first split the BAM into 1 BAM per Read Group. Then run bam2FastQ on each bam.'''
=== Output Files ===
By default, this program produces 3 output fastq files.
# unpaired reads
# first end of paired reads
# second end of paired reads
If the [[#Generate 1 Paired-End Output File (--merge)|<code>--merge</code>]] option is specified, the program produces 2 output fastq files.
# unpaired reads
# interleaved paired-end reads
The default fastq file names are determined by taking the base name of the input file and adding an extension for each filetype.
{|border="1" cellspacing="0" cellpadding="2"
! colspan="2"|Default !!colspan="2"|[[#Generate 1 Paired-End Output File (--merge)|<code>--merge</code>]]
|-
! Output File Contents !! Extension !! Output File Contents !! Extension
|-
|unpaired reads
| .fastq
|unpaired reads
| .fastq
|-
|first end of paired reads
| _1.fastq
| rowspan="2"|interleaved paired-end reads
(both first & second end)
| rowspan="2"|_interleaved.fastq
|-
|second end of paired reads
| _2.fastq
|}
If the inputFile was "myPath/myFile.bam", the resulting fastqs would be:
#myPath/myFile.fastq
#myPath/myFile_1.fastq
#myPath/myFile_2.fastq
With the [[#Generate 1 Paired-End Output File (--merge)|<code>--merge</code>]] option, the resulting fastqs would be:
#myPath/myFile.fastq
#myPath/myFile_interleaved.fastq
Instead of using the inputFile base name as the output file base, you can specify a different base name by using the [[#Output FastQ File Base Name (--outBase)|--outBase]] option.
You can optionally directly specify the output fastq filenames using:
* --firstOut firstReadInAPair.fastq (also used for the interleaved filename with [[#Generate 1 Paired-End Output File (--merge)|<code>--merge</code>]])
* --secondOut secondReadInAPair.fastq
* --unpairedOut unpairedReads.fastq
If any of these are not specified, the <code>--outBase</code> or default is used for that file.
= Usage =
./bam bam2FastQ --in <inputFile> [--readName] [--splitRG] [--qualField <tag>] [--refFile <referenceFile>] [--outBase <outputFileBase>] [--firstOut <1stReadInPairOutFile>] [--merge|--secondOut <2ndReadInPairOutFile>] [--unpairedOut <unpairedOutFile>] [--firstRNExt <firstInPairReadNameEx t>] [--secondRNExt <secondInPairReadNameExt>] [--rnPlus] [--noReverseComp] [--region <chr>[:<pos>[:<base>]]] [--gzip] [--noeof] [--params]
= Parameters =
<pre>
Required Parameters:
--in : the SAM/BAM file to convert to FastQ
Optional Parameters:
--readname : Process the BAM as readName sorted instead
of coordinate if the header does not indicate a sort order.
--splitRG : Split into RG specific fastqs.
--qualField : Use the base quality from the specified tag
rather than from the Quality field (default)
--merge : Generate 1 interleaved (merged) FASTQ for paired-ends (unpaired in a separate file)
use firstOut to override the filename of the interleaved file.
--refFile : Reference file for converting '=' in the sequence to the actual base
if '=' are found and the refFile is not specified, 'N' is written to the FASTQ
--firstRNExt : read name extension to use for first read in a pair
default is "/1"
--secondRNExt : read name extension to use for second read in a pair
default is "/2"
--rnPlus : Add the Read Name/extension to the '+' line of the fastq records
--noReverseComp : Do not reverse complement reads marked as reverse
--region : Only convert reads containing the specified region/nucleotide.
Position formatted as: chr:pos:base
pos (0-based) & base are optional.
--gzip : Compress the output FASTQ files using gzip
--noeof : Do not expect an EOF block on a bam file.
--params : Print the parameter settings to stderr
Optional OutputFile Names:
--outBase : Base output name for generated output files
--firstOut : Output name for the first in pair file
over-rides setting of outBase
--secondOut : Output name for the second in pair file
over-rides setting of outBase
--unpairedOut : Output name for unpaired reads
over-rides setting of outBase
</pre>
== Required Parameters ==
{{inBAMInputFile}}
== Optional Parameters ==
=== BAM File Is Sorted By Read Name (<code>--readname</code>) ===
The bam2FastQ program by default checks the sort order in the SAM/BAM header when converting to FASTQ, and if that is not specified, assumes it is sorted by coordinate.
To override the default and force it to assume the file is sorted by readname, specify the <code>--readName</code> option
The file does not need to be strictly sorted by read name. The only requirement is that matching read names are next to each other.
=== Split into RG Specific FASTQs (<code>--splitRG</code>) ===
Create RG specific FASTQ files.
Cannot be specified with firstOut/secondOut/unpairedOut since there will be a different filename for each RG.
Cannot write to stdout when <code>--splitRG</code> is specified.
Output filenames will be <outBase>.<RG>_1.fastq, <outBase>.<RG>_2.fastq, and <outBase>.<RG>.fastq. A fastq list file <outBase>.list will be created containing MERGE_NAME (the RG tag's SM value or outBase if the value is empty), fastq 1, fastq 2 (or . if it is a single ended fastq), and the RG tag string.
=== Use the Base Quality from the Specified Tag (<code>--qualField</code>) ===
By default, the quality field is used for the Base Qualities in the FASTQ file. Specify <code>--qualField <tagName></code> to use the base qualities from the specified tag instead of the quality field.
=== Generate 1 Paired-End Output File (<code>--merge</code>) ===
Use the <code>--merge</code> option to generate 1 interleaved (merged) FASTQ for paired-ends instead of 2 files. Unpaired reads are still written to a separate file.
The default extension for the output file is "_interleaved".
Use [[#Output FastQ File Name For the First End of Paired End (--firstOut)|<code>--firstOut</code>]] to override the filename of the interleaved file.
This parameter was added in version 1.0.10.
=== Reference File for Converting '=' in the Sequence to Bases (<code>--refFile</code>) ===
If the SAM/BAM file contains '=' in the sequence instead of the actual bases, the bam2FastQ program needs to convert the '=' back to the bases. To do that it needs the reference. Specify the reference by using <code>--refFile</code> followed by the reference filename.
For example:
./bam bam2FastQ --in myFile.bam --refFile myPath/myRefFile.fa
=== First in Pair FastQ ReadName Extension (<code>--firstRNExt</code>) ===
<code>--firstRNExt</code> overrides the default "/1" that is appended to the Read Name of the first-end of a read pair with the specified value.
=== Second in Pair FastQ ReadName Extension (<code>--secondRNExt</code>) ===
<code>--secondRNExt</code> overrides the default "/2" that is appended to the Read Name of the second-end of a read pair with the specified value.
=== Include the Read Name on the "+" line of the FASTQ (<code>--rnPlus</code>) ===
By default the read name is not included on the "+" line of the FASTQ files. To include the read name and the extension for paired-end reads, specify <code>--rnPlus</code>.
=== Do Not Reverse Complement Reverse Strands (<code>--noReverseComp</code>) ===
By default, reads marked as reverse in the BAM file are reverse complemented prior to writing to the FASTQ files. <code>--noReverseComp</code> disables this feature, and skips the reverse complement step.
For example, if a sequence is ACCGTG marked as reverse, the default FASTQ record will be written as: CACGGT
Specifying <code>--noReverseComp</code> would result in a FASTQ sequence of ACCGTG
=== Only Convert the Specified Region (<code>--region</code>) ===
Only convert reads containing the specified region/nucleotide.
Position formatted as: chr:pos:base
pos (0-based) & base are optional.
{{noeofBGZFParameter}}
{{paramsParameter}}
== Optional Output Filenames ==
=== Output FastQ File Base Name (<code>--outBase</code>) ===
You can replace the default output base name by using the <code>--outBase</code> option.
If the outBase was "myNewPath/myFastQBase", the resulting fastq's would be:
#myNewPath/myFastQBase.fastq
#myNewPath/myFastQBase_1.fastq
#myNewPath/myFastQBase_2.fastq
The value specified by this parameter is overridden by <code>--firstOut</code>, <code>--secondOut</code>, and <code>--unpairedOut</code>, but is used for whichever output files are not specified.
With the [[#Generate 1 Paired-End Output File (--merge)|<code>--merge</code>]] option, the resulting fastq's would instead be:
#myNewPath/myFastQBase.fastq
#myNewPath/myFastQBase_interleaved.fastq
=== Output FastQ File Name For the First End of Paired End (<code>--firstOut</code>) ===
This setting overides the default and <code>--outBase</code> file name.
The entire filename and extension must be specified.
Does not affect the filenames for the second end or for unpaired reads.
For example:
./bam bam2FastQ --in myFile.bam --firstOut myFileEnd1.fastq
=== Output FastQ File Name For the Second End of Paired End (<code>--secondOut</code>) ===
This setting overides the default and <code>--outBase</code> file name.
The entire filename and extension must be specified.
Does not affect the filenames for the first end or for unpaired reads.
For example:
./bam bam2FastQ --in myFile.bam --secondOut myFileEnd2.fastq
=== Output FastQ File Name For Unpaired Reads (<code>--unpairedOut</code>) ===
This setting overides the default and <code>--outBase</code> file names.
The entire filename and extension must be specified.
Does not affect the filenames for the paired-end fastq files.
For example:
./bam bam2FastQ --in myFile.bam --unpairedOut myFileUnpaired.fastq
{{PhoneHomeParameters}}
= Return Value =
Returns -1 if input parameters are invalid.
Returns the SamStatus for the reads/writes (0 on success, non-0 on failure).
