MAQ 参数说明.docx

MAQ 参数说明.docx

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MAQ参数说明

MAQKeyCommands

fasta2bfa

maqfasta2bfain.ref.fastaout.ref.bfa

ConvertsequencesinFASTAformattoMaq’sBFA（binaryFASTA）format.

fastq2bfq

maqfastq2bfq[-nnreads]in.read.fastqout.read.bfq|out.prefix

ConvertreadsinFASTQformattoMaq’sBFQ（binaryFASTQ）format.

OPTIONS:

-nINT

numberofreadsperfile[notspecified]

map

maqmap[-nnmis][-amaxins][-c][-1len1][-2len2][-dadap3][-mmutrate][-uunmapped][-emaxerr][-Mc|g][-N][-Hallhits][-Cmaxhits]out.aln.mapin.ref.bfain.read1.bfq[in.read2.bfq]2>out.map.log

Mapreadstothereferencesequences.

OPTIONS:

-nINT

Numberofmaximummismatchesthatcanalwaysbefound[2]

-aINT

Maximumouterdistanceforacorrectreadpair[250]

-AINT

MaximumouterdistanceoftwoRFpaiedread（0fordisable）[0]

-c

Mapreadsinthecolourspace（forSOLiDonly）

-1INT

Readlengthforthefirstread,0forauto[0]

-2INT

Readlengthforthesecondread,0forauto[0]

-mFLOAT

Mutationratebetweenthereferencesequencesandthereads[0.001]

-dFILE

Specifyafilecontainingasinglelineofthe3’-adaptersequence[null]

-uFILE

Dumpunmappedreadsandreadscontainingmorethannmismismatchestoaseparatefile[null]

-eINT

Thresholdonthesumofmismatchingbasequalities[70]

-HFILE

Dumpmultiple/all01-mismatchhitstoFILE[null]

-CINT

Maximumnumberofhitstooutput.Unlimitediflargerthan512.[250]

-Mc|g

methylationalignmentmode.AllC（orG）ontheforwardstrandwillbechangedtoT（orA）.Thisoptionisfortestingonly.

-N

storethemismatchpositionintheoutputfileout.aln.map.Whenthisoptionisinuse,themaximumallowedreadlengthis55bp.

NOTE:

*

Pairedendreadsshouldbepreparedintwofiles,oneforeachend,withreadsaresortedinthesameorder.Thismeansthek-threadinthefirstfileismatedwiththek-threadinthesecondfile.Thecorrespondingreadnamesmustbeidenticaluptothetailing‘/1’or‘/2’.Forexample,suchapairofreadnamesareallowed:

‘EAS1_1_5_100_200/1’and‘EAS1_1_5_100_200/2’.Thetailing‘/[12]’isusuallygeneratedbytheGAPipelinetodistinguishthetwoendsinapair.

*

Theoutputisacompressedbinaryfile.Itisaffectedbytheendianness.

*

Thebestwaytorunthiscommandistoprovideabout1to3millionreadsasinput.Morereadsconsumemorememory.

*

Option-ncontrolsthesensitivityofthealignment.Bydefault,ahitwithupto2mismatchescanbealwaysfound.Higher-nfindsmorehitsandalsoimprovestheaccuracyofmappingqualities.However,thisisdoneatthecostofspeed.

*

Alignmentswithmanyhigh-qualitymismatchesshouldbediscardedasfalsealignmentsorpossiblecontaminations.Thisbehaviouriscontrolledbyoption-e.The-ethresholdisonlycalculatedapproximatelybecausebasequalitiesaredividedby10atacertainstageofthealignment.The-Qoptionintheassemblecommandpreciselysetthethreshold.

*

ApairofreadsaresaidtobecorrectlypairedifandonlyiftheorientationisFRandtheouterdistanceofthepairisnolargerthanmaxins.Thereisnolimitontheminimuminsertsize.ThissettingisdeterminedbythepairedendalignmentalgorithmusedinMaq.Requiringaminimuminsertsizewillleadtosomewrongalignmentswithhighlyoverestimatedmappingqualities.

*

Currently,readpairsfromIllumina/Solexalong-insertlibraryhaveRFreadorientation.Themaximuminsertsizeissetbyoption-A.However,long-insertlibraryisalsomixedwithasmallfractionofshort-insertreadpairs.-ashouldalsobesetcorrectly.

*

Sometimes5’-endoreventheentire3’-adaptersequencemaybesequenced.Providing-drendersMaqtoeliminatetheadaptercontaminations.

*

Given2millionreadsasinput,maqusuallytakes800MBmemory.

mapmerge

maqmapmergeout.aln.mapin.aln1.mapin.aln2.map[...]

Mergeabatchofreadalignmentstogether.

NOTE:

*

Intheory,thiscommandcanmergeunlimitednumberofalignments.However,asmapmergewillbereadingalltheinputsatthesametime,itmayhitthelimitofthemaximumnumberofopeningfilessetbytheOS.Atpresent,thishastobemanuallysolvedbyendusers.

*

Commandmapmergecanbeusedtomergealignmentfileswithdifferentreadlengths.Allthesubsequentanalysesdonotassumefixedlengthanymore.

rmdup

maqrmdupout.rmdup.mapin.ori.map

Removepairswithidenticaloutercoordinates.Inprinciple,pairswithidenticaloutercoordinatesshouldhappenrarely.However,duetotheamplificationinsamplepreparation,thisoccursmuchmorefrequentlythanbychance.PracticalanalysesshowthatremovingduplicateshelpstoimprovetheoverallaccuracyofSNPcalling.

assemble

maqassemble[-sp][-mmaxmis][-Qmaxerr][-rhetrate][-tcoef][-qminQ][-NnHap]sin.ref.bfain.aln.map2>s.log

Calltheconsensussequencesfromreadmapping.

OPTIONS:

-tFLOAT

Errordependencycoefficient[0.93]

-rFLOAT

Fractionofheterozygotesamongallsites[0.001]

-s

Takesingleendmappingqualityasthefinalmappingquality;otherwisepairedendmappingqualitywillbeused

-p

Discardpairedendreadsthatarenotmappedincorrectpairs

-mINT

Maximumnumberofmismatchesallowedforareadtobeusedinconsensuscalling[7]

-QINT

Maximumallowedsumofqualityvaluesofmismatchedbases[60]

-qINT

Minimummappingqualityallowedforareadtobeusedinconsensuscalling[0]

-NINT

Numberofhaplotypesinthepool（>=2）[2]

NOTE:

*

Option-Qsetsalimitonthemaximumsumofmismatchingbasequalities.Readscontainingmanyhigh-qualitymismatchesshouldbediscarded.

*

Option-Nsetsthenumberofhaplotypesinapool.Itisdesignedforresequencingofsamplesbypoolingmultiplestrains/individualstogether.Fordiploidgenomeresequencing,thisoptionequals2.

indelpe

maqindelpein.ref.bfain.aln.map>out.indelpe

Callconsistentindelsfrompairedendreads.TheoutputisTABdelimitedwitheachlineconsistingofchromosome,startposition,typeoftheindel,numberofreadsacrosstheindel,sizeoftheindelandinserted/deletednucleotides（separatedbycolon）,numberofindelsonthereversestrand,numberofindelsontheforwardstrand,5’sequenceaheadoftheindel,3’sequencefollowingtheindel,numberofreadsalignedwithoutindelsandthreeadditionalcolumnsforfilters.

Atthe3rdcolumn,typeoftheindel,astarindicatestheindelisconfirmedbyreadsfrombothstrands,aplusmeanstheindelishitbyatleasttworeadsbutfromthesamestrand,aminusshowstheindelisonlyfoundononeread,andadotmeanstheindelistooclosetoanotherindelandisfilteredout.

Usersarerecommendedtorunthrough‘maq.plindelpe’tocorrectthenumberofreadsmappedwithoutindels.Formoredetails,seethe‘maq.plindelpe’section.

indelsoa

maqindelsoain.ref.bfain.aln.map>out.indelsoa

Callpotentialhomozygousindelsandbreakpointsbydetectingtheabnormalalignmentpatternaroundindelsandbreakpoints.TheoutputisalsoTABdelimitedwitheachlineconsistingofchromosome,approximatecoordinate,lengthoftheabnormalregion,numberofreadsmappedacrosstheposition,numberofreadsontheleft-handsideofthepositionandnumberofreadsontheright-handside.Thelastcolumncanbeignored.

Theoutputcontainsmanyfalsepositives.Arecommendedfiltercouldbe:

awk’$5+$6-$4>=3&&$4<=1’in.indelsoa

Notethatthiscommanddoesnotaimtobeanaccurateindeldetector,butmainlyhelpstoavoidsomefalsepositivesinsubstitutioncalling.Inaddition,itonlyworkswellgivendeepdepth（~40Xforexample）;otherwisethefalsenegativeratewouldbeveryhigh.

FormatConverting

sol2sanger

maqsol2sangerin.sol.fastqout.sanger.fastq

ConvertSolexaFASTQtostandard/SangerFASTQformat.

bfq2fastq

maqbfq2fastqin.read.bfqout.read.fastq

ConvertMaq’sBFQformattostandardFASTQformat.

mapass2maq

maqmapass2maqin.mapass2.mapout.maq.map

Convertobsoletemapass2’smapformattoMaq’smapformat.Theoldformatdoesnotcontainreadnames.

InformationExtracting

mapview

maqmapview[-bN]in.aln.map>out.aln.txt

Displaythereadalignmentinplaintext.ForreadsalignedbeforetheSmith-Watermanalignment,eachlineconsistsofreadname,chromosome,position,strand,insertsizefromtheoutercoorniatesofapair,pairedflag,mappingquality,single-endmappingquality,alternativemappingquality,numberofmismatchesofthebesthit,sumofqualitiesofmismatchedbasesofthebesthit,numberof0-mismatchhitsofthefirst24bp,numberof1-mismatchhitsofthefirst24bponthereference,lengthoftheread,readsequenceanditsquality.Alternativemappingqualityalwaysequalstomappingqualityifthereadsarenotpaired.Ifreadsarepaired,itequalstothesmallermappingqualityofthetwoends.Thisalternativemappingqualityisactuallythemappingqualityofanabnormalpair.

Thefifthcolumn,pairedflag,isabitwiseflag.Itslower4bitsgivetheorientation:

1standsforFF,2forFR,4forRF,and8forRR,whereFRmeansthatthereadwithsmallercoordinateisontheforwardstrand,anditsmateisonthereversestrand.OnlyFRisallowedforacorrectpair.Thehigherbitsofthisflaggivefurtherinformation.Ifthepairmeetsthepairedendrequirement,16willbeset.Ifthetworeadsaremappedtodifferentchromosomes,32willbeset.Ifoneofthetworeadscannotbemappedatall,64willbeset.Theflagforacorrectpairalwaysequalsto18.

ForreadsalignedbytheSmith-Watermanalignmentafterwards,