KeyCommandsfasta2bfamaqfasta2bfain.ref.fastaout.ref.bfa
Convert sequences in FASTA format to Maq's BFA (binary FASTA) format.
fastq2bfqmaqfastq2bfq [-nnreads] in.read.fastqout.read.bfq⎪out.prefix
Convert reads in FASTQ format to Maq's BFQ (binary FASTQ) format.
OPTIONS:-nINT number of reads per file [not specified]
mapmaqmap [-nnmis] [-amaxins] [-c] [-1len1] [-2len2] [-dadap3] [-mmutrate] [-uunmapped]
[-e maxerr] [-M c⎪g] [-N] [-Hallhits] [-Cmaxhits] out.aln.mapin.ref.bfain.read1.bfq
[in.read2.bfq] 2> out.map.log
Map reads to the reference sequences.
OPTIONS:-nINT Number of maximum mismatches that can always be found [2]
-aINT Maximum outer distance for a correct read pair [250]
-AINT Maximum outer distance of two RF paied read (0 for disable) [0]
-c Map reads in the colour space (for SOLiD only)
-1INT Read length for the first read, 0 for auto [0]
-2INT Read length for the second read, 0 for auto [0]
-mFLOAT Mutation rate between the reference sequences and the reads [0.001]
-dFILE Specify a file containing a single line of the 3'-adapter sequence [null]
-uFILE Dump unmapped reads and reads containing more than nmis mismatches to a separate file
[null]
-eINT Threshold on the sum of mismatching base qualities [70]
-HFILE Dump multiple/all 01-mismatch hits to FILE [null]
-CINT Maximum number of hits to output. Unlimited if larger than 512. [250]
-M c⎪g methylation alignment mode. All C (or G) on the forward strand will be changed to T
(or A). This option is for testing only.
-N store the mismatch position in the output file out.aln.map. When this option is in
use, the maximum allowed read length is 55bp.
NOTE:
* Paired end reads should be prepared in two files, one for each end, with reads are sorted in
the same order. This means the k-th read in the first file is mated with the k-th read in
the second file. The corresponding read names must be identical up to the tailing `/1' or
`/2'. For example, such a pair of read names are allowed: `EAS1_1_5_100_200/1' and
`EAS1_1_5_100_200/2'. The tailing `/[12]' is usually generated by the GAPipeline to
distinguish the two ends in a pair.
* The output is a compressed binary file. It is affected by the endianness.
* The best way to run this command is to provide about 1 to 3 million reads as input. More
reads consume more memory.
* Option -n controls the sensitivity of the alignment. By default, a hit with up to 2
mismatches can be always found. Higher -n finds more hits and also improves the accuracy of
mapping qualities. However, this is done at the cost of speed.
* Alignments with many high-quality mismatches should be discarded as false alignments or
possible contaminations. This behaviour is controlled by option -e. The -e threshold is only
calculated approximately because base qualities are divided by 10 at a certain stage of the
alignment. The -Q option in the assemble command precisely set the threshold.
* A pair of reads are said to be correctly paired if and only if the orientation is FR and the
outer distance of the pair is no larger than maxins. There is no limit on the minimum insert
size. This setting is determined by the paired end alignment algorithm used in Maq.
Requiring a minimum insert size will lead to some wrong alignments with highly overestimated
mapping qualities.
* Currently, read pairs from Illumina/Solexa long-insert library have RF read orientation. The
maximum insert size is set by option -A. However, long-insert library is also mixed with a
small fraction of short-insert read pairs. -a should also be set correctly.
* Sometimes 5'-end or even the entire 3'-adapter sequence may be sequenced. Providing -d
renders Maq to eliminate the adapter contaminations.
* Given 2 million reads as input, maq usually takes 800MB memory.
mapmergemaqmapmergeout.aln.mapin.aln1.mapin.aln2.map [...]
Merge a batch of read alignments together.
NOTE:
* In theory, this command can merge unlimited number of alignments. However, as mapmerge will
be reading all the inputs at the same time, it may hit the limit of the maximum number of
opening files set by the OS. At present, this has to be manually solved by endusers.
* Command mapmerge can be used to merge alignment files with different read lengths. All the
subsequent analyses do not assume fixed length any more.
rmdupmaqrmdupout.rmdup.mapin.ori.map
Remove pairs with identical outer coordinates. In principle, pairs with identical outer
coordinates should happen rarely. However, due to the amplification in sample preparation,
this occurs much more frequently than by chance. Practical analyses show that removing
duplicates helps to improve the overall accuracy of SNP calling.
assemblemaqassemble [-sp] [-mmaxmis] [-Qmaxerr] [-rhetrate] [-tcoef] [-qminQ] [-NnHap] out.cnsin.ref.bfain.aln.map 2> out.cns.log
Call the consensus sequences from read mapping.
OPTIONS:-tFLOAT Error dependency coefficient [0.93]
-rFLOAT Fraction of heterozygotes among all sites [0.001]
-s Take single end mapping quality as the final mapping quality; otherwise paired end
mapping quality will be used
-p Discard paired end reads that are not mapped in correct pairs
-mINT Maximum number of mismatches allowed for a read to be used in consensus calling [7]
-QINT Maximum allowed sum of quality values of mismatched bases [60]
-qINT Minimum mapping quality allowed for a read to be used in consensus calling [0]
-NINT Number of haplotypes in the pool (>=2) [2]
NOTE:
* Option -Q sets a limit on the maximum sum of mismatching base qualities. Reads containing
many high-quality mismatches should be discarded.
* Option -N sets the number of haplotypes in a pool. It is designed for resequencing of
samples by pooling multiple strains/individuals together. For diploid genome resequencing,
this option equals 2.
glfgenmaqglfgen [-sp] [-mmaxmis] [-Qmaxerr] [-rhetrate] [-tcoef] [-qminQ] [-NnHap] out.cnsin.ref.bfain.aln.map 2> out.cns.log
Calculate log-likelihood for all genotypes and store the results in GLF format (Genotyping
Likelihood Format). Please check MAQ website for detailed descriptions of the file format and
the related utilities.
indelpemaqindelpein.ref.bfain.aln.map > out.indelpe
Call consistent indels from paired end reads. The output is TAB delimited with each line
consisting of chromosome, start position, type of the indel, number of reads across the indel,
size of the indel and inserted/deleted nucleotides (separated by colon), number of indels on
the reverse strand, number of indels on the forward strand, 5' sequence ahead of the indel, 3'
sequence following the indel, number of reads aligned without indels and three additional
columns for filters.
At the 3rd column, type of the indel, a star indicates the indel is confirmed by reads from
both strands, a plus means the indel is hit by at least two reads but from the same strand, a
minus shows the indel is only found on one read, and a dot means the indel is too close to
another indel and is filtered out.
Users are recommended to run through `maq.pl indelpe' to correct the number of reads mapped
without indels. For more details, see the `maq.pl indelpe' section.
indelsoamaqindelsoain.ref.bfain.aln.map > out.indelsoa
Call potential homozygous indels and break points by detecting the abnormal alignment pattern
around indels and break points. The output is also TAB delimited with each line consisting of
chromosome, approximate coordinate, length of the abnormal region, number of reads mapped
across the position, number of reads on the left-hand side of the position and number of reads
on the right-hand side. The last column can be ignored.
The output contains many false positives. A recommended filter could be:
awk '$5+$6-$4 >= 3 && $4 <= 1' in.indelsoa
Note that this command does not aim to be an accurate indel detector, but mainly helps to
avoid some false positives in substitution calling. In addition, it only works well given deep
depth (~40X for example); otherwise the false negative rate would be very high.
FormatConvertingsol2sangermaqsol2sangerin.sol.fastqout.sanger.fastq
Convert Solexa FASTQ to standard/Sanger FASTQ format.
bfq2fastqmaqbfq2fastqin.read.bfqout.read.fastq
Convert Maq's BFQ format to standard FASTQ format.
mapass2maqmaqmapass2maqin.mapass2.mapout.maq.map
Convert obsolete mapass2's map format to Maq's map format. The old format does not contain
read names.
InformationExtractingmapviewmaqmapview [-bN] in.aln.map > out.aln.txt
Display the read alignment in plain text. For reads aligned before the Smith-Waterman
alignment, each line consists of read name, chromosome, position, strand, insert size from the
outer coorniates of a pair, paired flag, mapping quality, single-end mapping quality,
alternative mapping quality, number of mismatches of the best hit, sum of qualities of
mismatched bases of the best hit, number of 0-mismatch hits of the first 24bp, number of
1-mismatch hits of the first 24bp on the reference, length of the read, read sequence and its
quality. Alternative mapping quality always equals to mapping quality if the reads are not
paired. If reads are paired, it equals to the smaller mapping quality of the two ends. This
alternative mapping quality is actually the mapping quality of an abnormal pair.
The fifth column, paired flag, is a bitwise flag. Its lower 4 bits give the orientation: 1
stands for FF, 2 for FR, 4 for RF, and 8 for RR, where FR means that the read with smaller
coordinate is on the forward strand, and its mate is on the reverse strand. Only FR is allowed
for a correct pair. The higher bits of this flag give further information. If the pair meets
the paired end requirement, 16 will be set. If the two reads are mapped to different
chromosomes, 32 will be set. If one of the two reads cannot be mapped at all, 64 will be set.
The flag for a correct pair always equals to 18.
For reads aligned by the Smith-Waterman alignment afterwards, the flag is always 130. A line
consists of read name, chromosome, position, strand, insert size, flag (always 130), position
of the indel on the read (0 if no indel), length of the indels (positive for insertions and
negative for deletions), mapping quality of its mate, number of mismatches of the best hit,
sum of qualities of mismatched bases of the best hit, two zeros, length of the read, read
sequence and its quality. The mate of a 130-flagged read always gets a flag 18.
Flag 192 indicates that the read is not mapped but its mate is mapped. For such a read pair,
one read has flag 64 and the other has 192.
OPTIONS:-b do not display the read sequence and the quality
-N display the positions where mismatches occur. This flag only works with a .map file
generated by `maq map -N'.
mapcheckmaqmapcheck [-s] [-mmaxmis] [-qminQ] in.ref.bfain.aln.map > out.mapcheck
Read quality check. The mapcheck first reports the composition and the depth of the reference.
After that there is a form. The first column indicates the position on a read. Following four
columns which show the nucleotide composition, substitution rates between the reference and
reads will be given. These rates and the numbers in the following columns are scaled to 999
and rounded to nearest integer. The next group of columns show the distribution of base
qualities along the reads at a quality interval of 10. A decay in quality can usually be
observed, which means bases at the end of read are less accurate. The last group of columns
present the fraction of substitutions for read bases at a quality interval. This measures the
accuracy of base quality estimation. Idealy, we expect to see 1 in the 3? column, 10 in the 2?
column and 100 in the 1? column.
OPTIONS:-s Take single end mapping quality as the final mapping quality
-mINT Maximum number of mismatahces allowed for a read to be counted [4]
-qINT Minimum mapping quality allowed for a read to be counted [30]
pileupmaqpileup [-spvP] [-mmaxmis] [-Qmaxerr] [-qminQ] [-lsitefile] in.ref.bfain.aln.map >
out.pileup
Display the alignment in a `pileup' text format. Each line consists of chromosome, position,
reference base, depth and the bases on reads that cover this position. If -v is added on the
command line, base qualities and mapping qualities will be presented in the sixth and seventh
columns in order.
The fifth column always starts with `@'. In this column, read bases identical to the reference
are showed in comma `,' or dot `.', and read bases different from the reference in letters. A
comma or a upper case indicates that the base comes from a read aligned on the forward strand,
while a dot or a lower case on the reverse strand.
This command is for users who want to develop their own SNP callers.
OPTIONS:-s Take single end mapping quality as the final mapping quality
-p Discard paired end reads that are not mapped as correct pairs
-v Output verbose information including base qualities and mapping qualities
-mINT Maximum number of mismatches allowed for a read to be used [7]
-QINT Maximum allowed number of quality values of mismatches [60]
-qINT Minimum mapping quality allowed for a read to be used [0]
-lFILE File containing the sites at which pileup will be printed out. In this file the first
column gives the names of the reference and the second the coordinates. Additional
columns will be ignored. [null]
-P also output the base position on the read
cns2fqmaqcns2fq [-QminMapQ] [-nminNeiQ] [-dminDepth] [-DmaxDepth] in.cns > out.cns.fastq
Extract the consensus sequences in FASTQ format. In the sequence lines, bases in lower case
are essentially repeats or do not have sufficient coverage; bases in upper case indicate
regions where SNPs can be reliably called. In the quality lines, ASCII of a character minus 33
gives the PHRED quality.
OPTIONS:-QINT Minimum mapping quality [40]
-dINT Minimum read depth [3]
-nINT Minimum neighbouring quality [20]
-DINT Maximum read dpeth. >=255 for unlimited. [255]
cns2snpmaqcns2snpin.cns > out.snp
Extract SNP sites. Each line consists of chromosome, position, reference base, consensus base,
Phred-like consensus quality, read depth, the average number of hits of reads covering this
position, the highest mapping quality of the reads covering the position, the minimum
consensus quality in the 3bp flanking regions at each side of the site (6bp in total), the
second best call, log likelihood ratio of the second best and the third best call, and the
third best call.
The 5th column is the key criterion when you judge the reliability of a SNP. However, as this
quality is only calculated assuming site independency, you should also consider other columns
to get more accurate SNP calls. Script command `maq.plSNPfilter' is designed for this (see
below).
The 7th column implies whether the site falls in a repetitive region. If no read covering the
site can be mapped with high mapping quality, the flanking region is possibly repetitive or in
the lack of good reads. A SNP at such site is usually not reliable.
The 8th column roughly gives the copy number of the flanking region in the reference genome.
In most cases, this number approaches 1.00, which means the region is about unique. Sometimes
you may see non-zero read depth but 0.00 at the 7th column. This indicates that all the reads
covering the position have at least two mismatches. Maq only counts the number of 0- and
1-mismatch hits to the reference. This is due to a complex technical issue.
The 9th column gives the neighbouring quality. Filtering on this column is also required to
get reliable SNPs. This idea is inspired by NQS, although NQS is initially designed for a
single read instead of a consensus.
cns2viewmaqcns2viewin.cns > out.view
Show detailed information at all sites. The output format is identical to cns2snp report.
cns2refmaqcns2refin.cns > out.ref.fasta
Extract the reference sequence.
cns2winmaqcns2win [-wwinsize] [-cchr] [-bbegin] [-eend] [-qminQ] in.cns > out.win
Extract information averaged in a tilling window. The output is TAB delimited, which consists
of reference name, coordinate divided by 1,000,000, SNP rate, het rate, raw read depth, read
depth in approximately unique regions, the average number of hits of reads in the window and
percent GC.
OPTIONS:-wINT Size of a window [1000]
-cSTR Destinated reference sequence; otherwise all references will be used [null]
-bINT Start position, 0 for no constraint [0]
-eINT End position, 0 for no constraint [0]
-qINT Minimum consensus quality of the sites to be used [0]
SimulationRelatedfakemutmaqfakemut [-rmutrate] [-Rindelfrac] in.ref.fasta > out.fakeref.fasta 2> out.fake.snp
Randomly introduce substitutions and indels to the reference. Substitutions and sinlge base-
pair indels can be added.
OPTIONS:-rFLOAT Mutation rate [0.001]
-RFLOAT Fraction of mutations to be indels [0.1]
simutrainmaqsimutrainout.simupars.datin.read.fastq
Estimate/train parameters for read simulation.
simulatemaqsimulate [-dinsize] [-sstdev] [-NnReads] [-1readLen1] [-2readLen2] [-rmutRate] [-RindelFrac] [-h] out.read1.fastqout.read2.fastqin.ref.fastain.simupars.dat
Simulate paired end reads. File in.simupars.dat determines the read lengths and quality
distribution. It is generated from simutrain, or can be downloaded from Maq website. In the
output read files, a read name consists of the reference sequence name and the outer
coordinates of the pair of simulated reads. By default, simulate assumes reads come from a
diploid sequence which is generated by adding two different sets of mutations, including one
base-pair indels, to in.ref.fasta.
OPTIONS:-dINT mean of the outer distance of insert sizes [170]
-sINT standard deviation of insert sizes [20]
-NINT number of pairs of reads to be generated [1000000]
-1INT length of the first read [set by in.simupars.dat]
-2INT length of the second read [set by in.simupars.dat]
-rFLOAT mutation rate [0.001]
-RFLOAT fraction of 1bp indels [0.1]
-h add all mutations to in.ref.fasta and generate reads from the single mutated sequence
(haploid mode)
NOTE:
* Reads generated from this command are independent, which deviates from the truth. Whereas
alignment evaluation is less affected by this, evaluation on SNP calling should be performed
with caution. Error dependency may be one of the major causes of wrong SNP calls.
simustatmaqsimustatin.simu-aln.map > out.simustat
Evaluate mapping qualities from simulated reads.
SOLiDRelatedfasta2csfamaqfasta2csfain.nucl-ref.fasta > out.colour-ref.fasta
Convert nucleotide FASTA to colour-coded FASTA. Flag -c should be then applied to map command.
In the output, letter `A' stands for color 0, `C' for 1, `G' for 2 and `T' for 3. Each
sequence in the output is 1bp shorter than the input.
csmap2ntmaqcsmap2ntout.nt.mapin.ref.nt.bfain.cs.map
Convert color alignment to nucleotide alignment. The input in.ref.nt.bfa is the nucleotide
binary FASTA reference file. It must correspond to the original file from which the color
reference is converted. Nucleotide consensus can be called from the resultant alignment.
Miscellaneous/AdvancedCommandssubmapmaqsubmap [-qminMapQ] [-QmaxSumErr] [-mmaxMM] [-p] out.mapin.map
Filter bad alignments in in.map. Command-line options are described in the `assemble' command.
eland2maqmaqeland2maq [-qdefqual] out.mapin.listin.eland
Convert eland alignment to maq's .map format. File in.list consists of the sequence names that
appear at the seventh column of the eland alignment file in.eland and the name you expect to
see in maq alignment. The following is an example:
cX.fa chrX
c1.fa chr1
c2.fa chr2
If you are aligning reads in several batches using eland, it is important to use the same
in.list for the conversion. In addition, maq will load all the alignments and sort them in the
memory. If you have concatenate several eland outputs into one huge file, you should separate
it into smaller files to prevent maq from eating all your machine memory.
This command actually aims to show Eland alignment in Maqview. As no quality information is
available, the resultant maq alignment file should not be used to call consensus genotypes.
export2maqmaqexport2maq [-1read1len] [-2read2len] [-amaxdist] [-n] out.mapin.listin.export
Convert Illumina's Export format to Maq's .map format. Export format is a new alignment format
since SolexaPipeline-0.3.0 which also calculates mapping qualities like maq. The resultant
file can be used to call consensus genotypes as most of necessary information is available for
maq to do this accurately.
OPTIONS:-1INT Length of the first read [0]
-2INT Length of the second read [0]
-aINT Maximum outer distance for a correct read pair [250]
-n Retain filtered reads
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