Subread User Manual
Contents
1. i lt index gt Specify the base name of the index r lt input gt Give the name of an input file FASTQ FASTA format For paired end read data this gives the first read file and the other read file should be provided via the R option o lt output gt Give the name of the output file SAM format n lt int gt Specify the number of subreads extracted from each read 10 by default m lt int gt Specify the consensus threshold which is the minimal number of consensus subreads required for reporting a hit The consensus subreads are those subreads which vote for the same location in the reference genome for the read If pair end read data are provided this will be the consensus threshold for the read which receives more votes than the other read from the same pair 3 by default T lt int gt Specify the number of threads CPUs used for mapping 1 by de fault I lt int gt Specify the number of INDEL bases allowed in the mapping 5 by default P lt 3 6 gt Specify the format of Phred scores used in the input data 3 for phred 33 and 6 for phred 64 3 by default Output the uniquely mapped reads only Use mapping quality scores to break ties when more than one best mapping locations are found Use Hamming distance to break ties when more than one best map ping locations are found Indicate the discovered junction rea2. Subread User Manual Version 1 3 1 Wei Shi and Yang Liao Bioinformatics Division The Walter and Eliza Hall Institute of Medical Research The Department of Computer Science and Software Engineering The University of Melbourne Melbourne Australia 5 April 2013 1 Introduction Subread is a superfast sensitive and accurate read aligner which is designed to align reads generated from the 2nd and 3rd generation sequencers to a reference genome It supports a variety of sequencing platforms including Illumina GA HiSeq ABI SOLID Life Science 454 Helicos Heliscope and Ion Torrent It can align short reads long reads and reads of variable lengths Subread implements a mapping paradigm called seed and vote which is fundamen tally different from the mapping paradigm used by the current generation of read aligners The seed and vote paradigm extracts a number of subreads 16 mers from each read and then use these subreads to vote for the final location of the read rather than per forming a time consuming extension step Subread does not perform alignment for the read bases which are covered by the subreads that have made successful votes because no mismatches are allowed when mapping subreads to the reference genome Subread performs alignment for those bases which are not covered by the successfully mapped subreads and only align them to the reference bases located at the determined mapping location This makes Subread an e
3. ave a much smaller memory footprint Requesting less memory will increase running time C Build a color space index chri fa chr2 fa Give names of chromosome files 3 2 Aligning reads to the reference genome The subread align program extracts a number of subreads from each read and then uses these subreads to vote for the mapping location of the read The read bases which are not covered by the successfully mapped subreads will then be aligned to the reference bases located at the determined mapping location of the read Due to the flexible design of the seed and vote mapping paradigm subread align program can find mapping locations for the junction reads as well by using part of their sequences for mapping However subread align program can not determine either the precise position of the junction breakpoint in the read or the length of introns which the junction reads span These reads may be passed to the subjunc program to get the complete and accurate alignment information e g CIGAR information The subread align program allows up to 16 INDEL bases during the mapping for each read which is a lot more than other aligners A sophisticated algorithm was designed to determine the location and length of INDEL bases in the read in a more accurate manner Table 2 describes the arguments used by the subread align program Table 2 Arguments used by the subread align program Arguments Description
4. ctory located at the root directory Include the path to these variables to your environment variable PATH so that you can access them from anywhere Step 2 Build an index Build a base space index default subread buildindex o my_index chri fa chr2 fa Build a color space index subread buildindex c o my_index chri fa chr2 fa chri fa chr2 fa is a list of names of files which include chromosomal sequences It typically takes 1 hour to build an index for a human or mouse genome Step 3 Perform read alignment Use the following command to map single end reads subread align i my_index r reads txt o my_result sam reads txt is a FASTQ FASTA format file containing raw read data my_result sam is aSAM format file and contains mapping results including mapping locations CIGAR etc Use the following command to map paired end reads subread align i my_index r readsi txt R reads2 txt o my_result_PE sam Step 4 Discover exon exon junctions for RNA seq For single end data subjunc i my_index singleSAM my_result sam o my_junction_result For paired end data subjunc i my_index pairedSAM my_result_PE sam o my_junction_result Output of subjunc includes a BED file including discovered exon exon junctions and a SAM file including detailed mapping results eg CIGAR information for junction reads etc 3 Program description 3 1 Build an index for the reference genome The subread buildindex program buil
5. deletions The Smith Waterman algorithm is only applied for those reads which are found to contain insertions or deletions 2 by defaut E lt int gt Specify the penalty for extending the gap used by the Smith Waterman algorithm 0 by defaut X lt int gt Specify the penalty for mismatches used by the Smith Waterman algorithm 0 by defaut Y lt int gt Specify the score for matches used by the Smith Waterman algo rithm 2 by defaut 3 3 Finding exon junction locations The subjunc program maps junction reads by using a large number of subreads extracted from reads to discover the genomic regions spanned by these reads It then determines the precise spling points in the genome via donor acceptor site identification and sequence comparison Mapping location of each base in each read is recorded in the CIGAR field of the generate SAM file This program takes as input either a raw read data file FASTQ FASTA format or a SAM file e g SAM output from subread align and outputs discovered junction locations chromosomal locations in the reference genome and complete alignment in formation of the reads e g CIGAR information If SAM file is provided as input this program will use the potential junction reads marked by the aligner e g subread align to find junction locations and perform alignments for these reads to yield the CIGAR information If FASTQ FASTA file is provided the subread align progra
6. ds an base space or color space index using the reference sequences The reference sequences should be in FASTA format the header line for each chromosomal sequence starts with gt This program extracts all the 16 mer sequences from the reference genome at a 2bp interval and then uses them to build a hash table Keys in the hash table are unique 16 mers and values are their chromosomal locations By default the built index is parti tioned into blocks each of 3 7 GB in size and at any time only one block is present in the memory The index built for the human or mouse genome contains two trunks if default setting is used Table 1 describes the arguments used by the subread buildindex pro gram Table 1 Arguments used by the subread buildindex program Arguments Description 0 lt Specify the base name of the index to be created basename gt f lt int gt Specify the threshold for removing uninformative subreads highly repetitive 16mers Subreads will be excluded from the index if they occur more than threshold number of times in the reference genome Default value is 24 M lt int gt Specify the size of requested memory RAM in megabytes 8000MB by default With default value the entire built index will be loaded into memory in one go when carrying out read alignment which en ables the fastest mapping speed to be achieved The actual mem ory usage is 7600MB for mouse or human genome other species h
7. ds in the SAM output using S operation in the CIGAR string Subjunc program shall then be used to discover exon junction locations and to perform complete alignment for junction reads R lt input gt Provide the name of the second input file from paired end read data The program will then be switched to paired end read mapping mode p lt int gt Specify the consensus threshold for the read which receives less votes than the other read from the same pair This option is only applicable for mapping paired end read data The value of this recommended to be smaller than that of m option so as to rescue the poor quality reads by using the paired end distance information 1 by default d lt int gt Specify the minimum fragment template insert length for paired end read data 50 by default D lt int gt Specify the maximum fragment template insert length for paired end read data 600 by default Note that the read pairs which do not meet the distance criteria may still be reported provided that one or both reads can be successfully mapped S lt ff fr Specify the orientation of two reads from the same pair fr by rf gt default first read is located on forward strand and second read on reverse strand G lt int gt Specify the penalty for opening a gap when using the Smith Waterman dynamic programming algorithm to detection insertions and
8. en be switched to paired end read mapping mode d lt int gt Specify the minimum fragment template insert length for paired end read data 50 by default D lt int gt Specify the maximum fragment template insert length for paired end read data 600 by default Note that the read pairs which do not meet the distance criteria may still be reported provided that one or both reads can be successfully mapped S lt ff fr Specify the orientation of two reads from the same pair fr by ey gt default first read is located on forward strand and second read on reverse strand 4 Citation Yang Liao Gordon K Smyth and Wei Shi 2013 The Subread aligner fast accu rate and scalable read mapping by seed and vote Nucleic Acids Research Volume 41 Advance Access April 4 Epub ahead of print http nar oxfordjournals org content early 2013 04 03 nar gkt214 abstract 5 Contact Please email shi wehi edu au or liao wehi edu au for any inquires
9. m will be called to carry out the alignemnt Table 3 describes the arguments used by the subjunc program Table 3 Arguments used by the subjunc program Arguments Description i lt index gt Specify the base name of the index r lt input gt Give the name of an input file FASTQ FASTA format Both base space and color space read data are supported For paired end read data this gives the first read file and the other read file should be provided via the R option subread align program will be used to align the reads o lt output gt Give the name of the output file n lt int gt Give the number of subreads extracted from each read 14 by de fault singleS AM Use as input file a SAM file which includes mapping results for lt input gt single end reads e g subread align output pairedSAM lt Use as input file a SAM file which includes mapping results for input gt paired end reads e g subread align output T lt int gt Specify the number of threads CPUs used for mapping 1 by de fault I lt int gt Specify the number of INDEL bases allowed in the mapping 5 by default P lt 3 6 gt Specify the format of Phred scores used in the input data 3 for phred 33 and 6 for phred 64 3 by default R lt input gt Provide the name of the second input file in paired end data The program will th
10. xtremely fast aligner It is 2 40 times faster than others Subread achieves high mapping speed without losing sensitivity and accuracy In fact it is much more sensitive than other aligners for mapping RNA seq data because of its highly flexible design Moreover Subread has superior accuracy in finding INDEL bases in the reads and it can find up to 16 inserted or deleted bases The subjunc program included in the subread package can be used to find exon junc tion location for RNA seq data or gene fusion locations for RNA seq and gDNA seq data It returns a BED file which includes the chromosomal locations of exon junctions and or gene fusions and a SAM file which records the detailed mapping information for each read e g CIGAR information Subread is implemented in C programming language An R package called Rsub read which provides wrapper functions for the C function can be downloaded from http bioconductor org packages 2 10 bioc html Rsubread html 2 Quick start A base space single end read dataset can be mapped by following the steps below Step 1 Install Subread On a UNIX computer uncompress the downloaded subread 1 x x tar gz file Change to the root directory of this package enter the src subdirectory and type make command This will compile the source code and create several executables including subread buildindex subread align and subjunc These executables will be automatically moved into the bin subdire
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