brat user manual 1 2 1
Contents
1. PCR read 1 maps to DS rev compl of read 2 maps to DS equivalentrly read 2 maps to DS A at ciT elG lc t T ale t c Il 5 aAlalc 7 r v a E G 3 PCR rev compl of read 1 maps to DS equivalentrly read 1 maps to DS read 2 maps to DS AACCTIAG lc t tlale cla 5 AAC 1 ON OE 3 PCR rev compl of read 1 maps to DS equivalentrly read 1 maps to DS read 2 maps to DS AATCTIGG c T T A G T G saa cira gt PCR read 1 maps to DS rev compl of read 2 maps to DS equivalentrly read 2 maps to DS AA jcj r rjale cla s aja Si aside ic PCR re read 2 PCR read 1 read 1 A enriched G depleted PCR read 2 PCR re read 1 read 1 A enriched G depleted PCR read 2 PCR re read 1 read 1 T enriched C depleted PCR rc read 2 PCR read 1 read 1 T enriched C depleted 5 Ds Figure 3 Here we show mapping of the pairs sequenced from all four strands The bottom strand shows positive strand of the reference with methylated cytocine shown in blue and methylated cytocine on negative strand shown as G in orange This figure demonstrates that if we use simplified approach of counting methylated level of cytocines from positive and negative strands then we introduce a bias in counting methylation level of unmethylated cytocines either cytocines2. CTT G GIAA Cc p _ GAJA Ls 3 GIAA Cm 3 C A GAAT T A sequenced pairs PCR AATCTGG PCR TGCTAAG PCR CACTAAG PCR AACCTAG CACTAAG AACCTAG AATCTGG TGCTAAG Figure 2 Here we show the sequenced pairs resulted from sequencing all four PCR product strands each of which serves as a template during sequencing If PCR1 is attached to the flow cell then in single end sequencing 5 end of this strand is sequenced in paired end sequencing 5 end of PCR1 is sequenced then PCR1 is copied into its reverse complement PCR1 and 5 end of PCR1 is sequenced as the second mate of a pair Similarly sequencing produces reads for the rest of strands If procedure has some technical details that ensure that only original genomic strands serve as templates for sequencing then even though we have four PCR product strands we ll have reads sequenced only from PCR1 and PCR2 with mates ordering shown above in paired end sequencing the left column of reads are mates 1 will have reads IDs end with 1 and will be in one file the right column of reads are mates 2 will have reads IDs end with 2 and will be in the other file of Illumina s sequenced reads
3. format NOTE current version of BRAT and BRAT large do NOT support FASTQ format These additional files are for users to track original reads names and corresponding base quality scores Commands to run brat and brat large BRAT and BRAT large map raw reads output from trim to references that have to be in FASTA format one reference per FASTA file BRAT accepts a single file that contains names of FASTA files with the references To map bisulfite single end reads run either of the commands brat r_ references_names txt s prefix_readsi txt bs o output_results txt brat large r references_names txt s prefix_readsi txt bs o output_results txt brat r_ references_names txt s prefix_reads2 txt bs o output_results txt A brat large r references_names txt s prefix_reads2 txt bs o output_results txt A The file references_names txt contains the names of the FASTA files with the references The file output_results txt contains the results of the mapping only uniquely mapped reads are in this file The option bs specifies that mapping is for bisulfite sequenced reads To map normal not bisulfite treated reads run similar commands but without bs option brat r references_names txt s prefix_reads1 txt o output_results txt brat large r references_names txt s prefix_readsi txt o output_results txt To map bisulfite paired end reads run either of the following commands brat r references_names txt 1 prefix
4. that are partially methylated across cells of a sample or completely unmethylated For example read 2 mate 2 from PCR1 maps to negative strand its reverse complement maps to positive strand and therefore initially we contributed ACGT counts from this read toward negative strand This was incorrect in this Figure bias is introduced by counting G from this read in red circle toward unmethylated G i e unmethylated cytocine on negative strand The count of another G from this read in green circle toward methylated G did not introduce a bias since methylated G has only Gs mapped to it but the coverage of methylated Gs is also not what it would have been were we using the correct counting Similar bias is introduced when counting contribution of ACGT from mates 2 from PCR2 From Figures 1 3 we can observe that mate 2 of PCR1 strand is reverse complement 3 end of PCR1 thus mate 2 must reflect methylation on positive strand rather than on negative strand For example methylated C on PCR1 will be G on PCR1 and unmethylated C which is T on PCR1 will be A on PCR1 Thus T C and C C where T and C respectively belong to PCR1 must contribute to the count of methylation level of cytocines on positive strand Similarly A G and G gt G with A and G belonging to PCR2 must contribute to the count of methylation level of cytocines on negative strand These changes have been made to acgt count and now ACGT counting from s
5. that removes copy duplicates keeping only a randomly chosen one where copy duplicates are the reads that are mapped to the same start position in the reference Finally acgt count processes all the mapped results to produce methylome a map with methylation status of each cytocine Tool check strands is used for information on ACGT distribution in the reference and input reads 2 SYSTEM AND SPACE REQUIREMENTS BRAT stands for Bisulfite treated Reads Analysis Tool There are two versions of BRAT BRAT and BRAT arge Both programs run on 64 bit architecture under Linux Unix operating system Both versions work with reads of lengths 24 bases and above BRAT accepts up to 4 2G references with total size of references up to 4 2G base pairs BRAT large can work with up to 4 2G references with the size of the largest reference up to 4 2G base pairs BRAT works best with relatively small genomes because it uses significantly more space than BRAT large BRAT is faster than BRAT large that maps reads to the references sequentially it maps all reads to the first reference then all reads to the second reference and so on The space requirement of BRAT large depends on the size of the largest reference in the set of input references whereas BRAT s space requirement depends on the total size of all input references measured in base pairs Let N be the total size of a genome in base pairs T be the size of the largest reference in base pairs and R be the to
6. with trimmed reads To trim paired end reads in the files reads1 fastq and reads2 fastq in FASTQ format run the command trim 1 readsi fastq 2 reads2 fastq P prefix q 20 L 64 m 2 Here we assume that reads1 fastq contains sequenced 5 mates and reads2 fastq contains sequenced 3 mates The output will be in four files with raw reads prefix_readsI txt prefix_reads2 txt prefix_matesl txt and prefix_mates2 txt To further map paired end reads use prefix_reads1 txt and prefix_reads2 txt as input files for paired end mapping with brat or brat large The file prefix_mates txt contains reads from the file reads1 fastq whose mates have shorter length than 24 bases after trimming Similarly the file prefix_mates2 txt contains reads from the file reads2 fastq whose mates are shorter than 24 bases The user can further map these files prefix_mates txt and prefix_mates2 txt as single end reads for BS mapping of the reads in prefix_mates2 txt the user must specify A option for mapping to work correctly the same is true if a user wishes to map the reads in prefix_reads2 txt as single reads Additional output files are prefix_pairl fastq prefix_pair2 fastq prefix_mateslfastq and prefix_mates2 fastq These files have the same reads as do files prefix_reads1 txt prefix_reads2 txt prefix_mates1 txt and prefix_mates2 txt respectively except the files prefix_pairl fastq prefix_pair2 fastq prefix_mates1 fastq and prefix_mates2 fastq are in FASTQ
7. 1 3 below s faa lc le t elr ra elce z H ei Ps DH sal s r r e a c c _e a a T eG c 5 add adaptors perfor bisulfite treatment s fala u c z e e e e u G st t a a c c e aa tc alc 5 PC s 2 a a c c T A _ c T T A G C A 3 PCRS s afr releja t c e ala z c e z Figure 1 After the special adapters with methylated cytokines are ligated to DNA fragments sodium bisulfite treatment BS treatment is applied to DNA fragments after which unmethylated cytocines are converted to uracils and later to thymines during PCR for library amplification Note that after BS treatment there are four distinct PCR product strands PCR1 and PCR2 correspond to original genomic strands and PCR1 and PCR2 the reverse complements of PCR1 and PCR2 respectively Note that PCR1 and PCR2 are T rich and C depleted since unmethylated cytocines converted to thymines and PCR1 and PCR2 are A rich and G depleted as the reverse complements of PCR1 and PCR2 respectively 5 A A T C T G G PCR 3 TT GGATIC Eae a 5 AACCTAG V PCR 3 AIGJAIC C 5 AJA C GIG
8. C i 3 G i 4 G and i 5 T The second choice If a user provides option B acgt count r references_names txt P prefix p pairs_results txt s single_results txt B then the output is in two files one file for positive strand and another for negative strand output files will contain words forw and rev to distinguish between strands Each line in the output corresponds to a base in the genome that is either a cytocine on positive strand or cytocine on negative strand given in separate files Output format chrom start end total methylation_level strand where chrom is the reference name start and end are positions in the genome Note base count in a reference starts with 0 total takes one of the values CHH X CHG X or CpG X where X is the sum of counts of Cs and Ts mapped to this base methylation level is calculated as the number of Cs over the total methylation level count_C count_C count_T CHH CHG and CpG describe the sequence content following C if two consecutive bases that follow C are not G then total CHH X if the first consecutive base following C is non G and the second consecutive is G then total CHG X and finally if G follows C we have CG di nucleotide then total CpG X Output format for trim The tool trim accepts FASTQ files with reads pairs as input trims the ends of the reads whose base quality scores are lower than the user specified threshold or whose ends are Ns The outpu
9. S mismatches within the first X bases and any number of non BS mismatches in the other bases of reads The package includes three additional tools trim brat large build and acgt count The latter aligns mapped reads to the genome and counts mapped nucleotides at each base for forward and reverse strands separately The tool trim accepts FASTQ files with reads pairs as input trims the ends of the reads whose base quality scores are lower than the user specified threshold and trims Ns at the ends of the reads The tool brat large build is used with option P with BRAT arge 3 COMMANDS AND INPUT To uncompress run tar zxvf brat 1 2 tar gz To build cd brat 1 2 make This will create executable programs brat large brat large build acgt count trim rev compl check strands and remove dupl Input format of the reads for BRAT and BRAT large is raw reads e Read lt string gt a read after using trim e Start lt int gt the number of bases trimmed at the beginning of the original read e End lt int gt the number of bases trimmed at the end of the original read To convert reads from FASTQ format to raw reads one should run trim If a user does not wish to trim reads low quality score bases then he she should omit the option for the base quality score threshold the default threshold equals to zero so all reads will be in the output without change in lengths except for reads having Ns at the ends If reads have Ns at the en
10. USING BRAT 1 2 1 This new version has a different input and output formats to enable BRAT to identify copy duplicates the reads mapped to the same location in the genome A new tool remove dupl chooses and keeps a randomly chosen representative of copy duplicates and removes the rest of copy duplicates 1 ANALYSIS PIPELINE Currently BRAT includes the following tools brat brat large brat large build acgt count trim rev compl check strands and remove dupl The flow of the analysis pipeline is given in the figure below Reads in References FASTQ in FASTA Vv check mim strands AA brat or brat large Mapped gt Unmapped a results d lt reads d A N SS only from 2 original X genomic strands _ Ure reads zara gt a va Yo remove No dupl rev compl acgt count brat or brat large First tool trim takes reads in FASTQ format trims low quality bases from both ends as well as Ns and outputs reads in raw reads format that are accepted by brat and brat large After trim brat or brat large are used to map the reads to the reference genome If the reads are sequenced from four PCR product strands then rev compl is used to take reverse complements of the unmapped reads followed by mapping of the results with brat or brat large Next mapped results are used as input for remove dupl
11. _reads1 txt 2 prefix_reads2 txt pe bs o output_results txt brat large r references_names txt 1 prefix_readsi txt 2 prefix_reads2 txt pe bs o output_results txt The option pe specifies paired end mapping and as with single end reads bs specifies bisulfite mapping The results of the mapping will be in output_results txt One can choose to pre build genome index first and then use brat large on this index to speed up mapping Use option P and tool brat large build brat large build r _references_names txt P some_directory options S bs f brat large P some_directory 1 prefix_readsi txt 2 prefix_reads2 txt pe o output_results txt options To use this option that allows to separate hashing of the genome from mapping of reads one has to create a directory some_directory the name of a directory in the example above and to run the following commands 1 to build a hashing index and 2 to map reads If the directory some_directory is the directory inside the directory in which you run the commands above then providing the name some_directory is sufficient However if you run these commands in the directory that does not have some_directory in it then please provide a full path to some_directory The tool brat large build will output hashing index of the references into some_directory For human genome space on hard disk for hashing index is 15GB and 26GB when option S is used The command for tool brat large with op
12. assuming prefix_readsI txt was provided in the command line with option s for single reads and with paired end reads in prefix_reads1 txt unm and _ prefix_reads2 txt unm files if prefix_readsI txt and prefix_reads2 txt were used with the options 1 and 2 in the corresponding command line Unmapped reads in the output files are in raw reads format lt read X Y gt where X and Y are the number of bases trimmed from the beginning and end of original read respectively there are no ambiguous reads pairs in the files with unmapped reads s lt single end reads file gt to specify the file with input reads for single end reads mapping 1 lt paired end reads file gt to specify the file with 5 mates for paired end reads mapping in our example prefix_reads1 txt This option is also used with acgt count 2 lt paired end reads file gt to specify the file with 3 mates for paired end reads mapping in our example above prefix_reads2 txt This option is also used with acgt count pe to specify paired end reads mapping default is false i e single end mapping bs to specify bisulfite sequenced reads mapping Without this option mapping will be done as for normal not bisulfite treated sequenced reads i lt positive integer gt to specify minimum insert size for paired end mapping the minimum distance allowed between the leftmost ends of the mapped mates on forward strand default is 100 a lt positive integer gt to specif
13. ded The files whose names are listed in the files pairs_results txt and single_results txt must be in BRAT s output format To make this point clear assume a user ran brat on paired end reads and had the output file with the results in output_pairs_results txt to run acgt count the user must store the name of this file in pairs_results txt file and run acgt_count using pairs_results txt i e pairs_results txt will have in this case one line namely output_pairs_results txt The command for this example is acgt count r references_names txt P prefix p pairs_results txt Please note that if a user has paired end reads files with mates 1 and mates 2 and wishes to map the mates as single end reads then the user must provide names of the files with results for mates 1 and mates 2 separately using options 1 and 2 This will ensure unbiased ACGT counting when reads are sequenced from two original genomic strands acgt count r references_names txt P prefix p pairs_results txt 1 single_results_mates1 txt 2 single_results_mates2 txt To produce a more concise output use option B choose an appropriate command from the commands below acgt count r references_names txt P prefix p pairs_results txt s single_results txt B acgt count r references_names txt P prefix p pairs_results txt 1 single_results_matesi1 txt 2 single_results_mates2 txt B This program takes care of overlapping mates if two mates of a pair overlap the
14. ds trim trims Ns at the ends and outputs only those reads whose length after trimming is greater or equal to 24 bases A command to run trim This program trims low quality bases lower than a threshold given with option g and Ns from each end of a read bases are trimmed one at a time from both ends of a read until a base with quality score greater or equal than q is encountered similarly all consecutive Ns from both ends of a read are trimmed This tool outputs only those reads whose length is at least 24 after trimming and that have at most m internal Ns the number of allowable internal Ns is set by option m To trim single end reads in the file reads fastq in FASTQ format and output trimmed raw reads into a file with name prefix_reads1 txt ran the command trim s reads fastq P prefix q 20 L 64 m 2 This will trim bases whose base quality scores are lower than 20 from the ends of reads The option L specifies the smallest value of the range of base quality scores in ASCII representation please see Commands Options for details To learn more about Phred scores please visit http www phrap com phred Option m allows each read having at most 2 internal Ns Option P provides prefix to the output file names it might contain a path for an output file P home directory prefix If the user does not wish to trim ends with low base quality scores the q option is not specified For single end reads there is a single output file
15. e option L uses the values in the Smallest Value in ASCII representation column Type Smallest Score Largest Score Smallest Value in Largest Value in ASCII representation ASCII representation Phred quality score Sanger format o 23 oe 126 Solexa Illumina 1 0 5 62 59 126 Soloexa Illumina 0 62 64 126 1 3 B specifies the second option for output with acgt count please read Output format for acgt count A command to run remove dupl This program processes the mapping results and removes copy duplicates it outputs all reads that are mapped to a unique genomic location and only a randomly chosen one out of copy duplicates the reads mapped to the same location remove dupl r references_names txt p pairs_results txt 1 single_results_mates1 txt 2 single_results_mates2 txt NOTE the file pairs_results txt does not contain the actual results it contains the names of the files with the results for paired end mapping and similarly single_results txt file contains the names of the files with the actual results for single end mapping For example the content of pairs_results txt is output_pairs_lanel txt output_pairs_lane2 txt and the content of single_results_mates1 txt is output_singles_mates1_lane1 txt output_singles_mates1_lane2 txt and the content of single_results_mates2 txt is output_singles_mates2_lane1 txt output_singles_mates2_lane2 txt The output of remove dupl a
16. e mails concerned that the sequenced reads must be products of sequencing of all four strands The best way to be sure how many strands have been sequenced is talking to Illumina tech support but there is also a quick and dirty method to answer this question This approach will work best with genomes whose ACGT distribution is close to uniform or with genomes that CG rich We cannot be positive that the way we will explain shortly can be successfully applied to AT rich genomes We added a new tool check strands that calculates ACGT distribution in the genome and in mates 1 and mates 2 of paired end reads For the case when pairs are sequenced only from two original genomic strands you should notice significant increase of Ts in mates 1 and decrease of Cs compared to Ts and Cs in the genome and significant increase of As in mates 2 and consequently decrease of Gs compared to As and Gs in the genome If four strands are sequenced then the file with mates 1 contains a mixture of reads sequenced from original genomic strands as well as their reverse complements Thus the increase of both As and Ts and decrease of counts of Cs and Gs should be observed in both files with mates 1 and mates2 This conclusion is derived from the fact that after BS treatment original genomic strands are T rich and C depleted and consequently their reverse complements PCR1 and PCR2 are A rich and G depleted Below are the commands to run check strands Single end r
17. eads check strands r references_names txt s readsi fastq o output_results txt Paired end reads check strands r references_names txt 1 readsi fastq 2 reads2 fastq o output_results txt Here reference_names txt as before it is the file with names of files in FASTA with references reads1 fastq and reads2 fastq are FASTQ files from Illumina with sequenced reads for paired end reads with mates 1 and mates 2 The output is in output_results txt it is self explanatory with proportions of A C G and T in that order in references in reads from reads 1 fastq and in reads from reads2 fastq How to use BRAT with reads sequenced from four strands For single end reads if reads are sequenced from four PCR product strands namely PCR1 PCR2 PCR1 and PCR2 then run BRAT as explained before with option u that will output unmapped reads into a separate file say unmapped txt Then use our new tool rev compl with unmapped txt as follows rev compl s unmapped txt o rev_compl_of_unmapped txt The output file rev_compl_of_unmapped txt will contain reverse complement of reads from unmapped txt file Then map reads from rev_compl_of_unmapped txt as usual see how to map single end reads with BRAT For paired end reads map reads as usual but use u option to output unmapped reads use appropriate command of these two brat r references_names txt 1 prefix_reads1 txt 2 prefix_reads2 txt pe bs o output_results txt u brat
18. econd mates of pairs sequenced from PCR1 and PCR2 is done without bias which is demonstrated in Figure 4 How to avoid the counting bias PCR re read 2 c Alicia PCR read 1 c Alcic PCR read 2 T Giciec PCR rec read 1 T G C G PCR read 2 CJAC IG PCR rc read 1 CY AJC PCR re read 2 GICKG PCR read 1 TJGKC G 5 Ds sal 7 PCR re read 2 CIA CIG PCR read 1 caicia PCR read 2 T GICIG PCR rc read 1 T GIC G PCR read 2 C a che PCR re read 1 a cke PCR re read 2 TI G G PCR read 1 N G os LS Figure 4 If mate 1 of a pair maps to positive strand case of a pair sequenced from PCR1 then we increment ACGT count from both mates for positive strand and methylation level of a cytocine on the positive strand can be measured as total Cs mapped to positive strand to the sum of total Cs and total Ts mapped to the positive strand If mate 1 of a pair maps to negative strand case of a pair sequenced from PCR2 then we increment ACGT count from both mates for negative strand Methylation level can be determined for each G on positive strand of the reference G on positive strand corresponds to C on negative strand by looking into acgt count output for negative strand and taking the ratio of total Gs mapped to this position and the sum of total Gs and As mapped to this position We received a couple of
19. he total of allowed non BS mismatches then some of the pairs from PCR1 and PCR2 can be mapped together with the paired end reads sequenced from the original strands inducing ACGT count bias showed in Figure 5 BRAT does not have any counting bias for the case when only original strands are sequenced however in case of four strands being sequenced there might be a bias that users must account for by some other means statistics etc This is shown in Figure 5 PCR read 1 PCR read 2 PCR re read 1 PCR read 2 PCR re read 1 PCR rc read 2 PCR read 1 5 DS Figure 5 There is no acgt count bias for the case when two original strands are sequenced but there might be a bias from mapped paired end reads sequenced from PCR1 and PCR2 strands for unmethylated cytocines or for partially methylated cytocines if the mates have no BS mismatches or the number of inappropriate see above BS mismatches is less than or equal to the number of allowed non BS mismatches
20. l position is 10 2 8 If the number of trimmed bases at the end of a read was 3 and the reverse complement of the read is mapped to the position 10 on positive strand then original position position 3 10 3 7 The original positions are used to identify copy duplicates Output format for brat and brat large for paired end mapping Read id lt integer gt a consecutive number of a read in the reads input file that starts with 0 Read 1 lt string gt the first mate of a pair given as in the input file prefix_reads1 txt Read 2 lt string gt the second mate of a pair given as in the input file prefix_reads2 txt Reference name lt string gt a name of the reference to which the pair is mapped the first word following gt in a FASTA file e Strand if 5 mate from prefix_reads1 txt is mapped to forward strand consecutively 3 mate from prefix_reads2 txt is mapped to reverse strand and if the 5 mate is mapped to reverse strand and 3 mate to forward strand e Position 1 lt integer gt position within the reference starting with 0 where 5 mate is mapped the leftmost position on forward strand e Position 2 lt integer gt position within the reference starting with 0 where 3 mate is mapped the leftmost position on forward strand The number of non BS mismatches lt integer gt the number of mismatches in the alignment for 5 mate e The number of non BS mismatche
21. large r references_names txt 1 prefix_reads1 txt 2 prefix_reads2 txt pe bs o output_results txt u The unmapped reads from prefix_reads1 txt will be in prefix_reads1 txt unm and unmapped reads from prefix_reads2 txt will be in prefix_reads2 txt unm Then take reverse complements of unmapped reads please provide output files with option o rev compl s prefix_reads1 txt unm o rc_ prefix_reads1 txt unm rev compl s prefix_reads2 txt unm o rc_prefix_reads2 txt unm Then run BRAT again on reverse complements of unmapped reads and use option A use either of the commands below brat r references_names txt 1 rc_unm_prefix_reads1 txt 2 rc_unm_prefix_reads2 txt o output_results2 txt A brat large r references_names txt 1 rc_unm_prefix_reads1 txt 2 rc_unm_prefix_reads2 txt o output_results2 txt A The rest of the tools are used as before Why does this work BRAT allows only T C BS mismatches if mate 1 maps to positive strand and only A G BS mismatches if reverse complement of mate 1 maps to positive strand Thus when we first time map paired end reads only pairs sequenced from the original strands are mapped after we take reverse complement then mapping is done for the pairs sequenced from PCR1 and PCR2 However if a read does not have BS mismatches or the number of inappropriate BS mismatches A G in case when mate 1 maps to positive strand and T C when reverse complemet of mate 1 maps to positive strand is less than t
22. n ACGT count is done only for one mate in the overlapped region This program also takes care of producing un biased ACGT count from mates 2 3 mates Please see Details on ACGT count section for details A command to run rev compl Please read subsection How to use BRAT with reads sequenced from four strands A command to run check strands Please read section Details on ACGT count 4 OUTPUT FORMAT Output format for brat and brat large for single end mapping Read id lt integer gt a consecutive number of a read in the reads input file that starts with 0 Read 1 lt string gt the read given as in the input file prefix_reads1 txt e Reference name lt string gt a name of a reference to which the read is mapped the first word following gt in a FASTA file Strand if the read is mapped to forward strand and if the read is mapped to reverse strand e Position lt integer gt position within the reference starting with 0 where the read is mapped the leftmost position on forward strand The number of non BS mismatches lt int gt Original position lt integer gt position within the reference starting with 0 where the original read is mapped the leftmost position on forward strand where original read is the read before its ends have been trimmed For example if the number of trimmed bases at the beginning of a read is 2 and the read is mapped to positive strand at the position 10 then origina
23. re the files with the same names as before with additional extension nodup output_pairs_lanel txt nodupl output_pairs_lane2 txt nodupl output_singles_mates1_lane1 txt nodupl output_singles_mates1_lane2 txt nodupl output_singles_mates2_lane1 txt nodupl output_singles_mates2_lane2 txt nodupl For single end mapping run the following command remove dupl r references_names txt s single_results txt The file single_results txt contains the names of the files with mapping results Please note that the output files with extension nodupl are opened with C app option opens a file and appends output to the file s content This means that once you have run remove dupl you will have nodupl files and if you want for some reason to run remove dupl on the same files and possibly some additional files you need to remove nodup files for the corresponding files first and only then re run remove dupl For example as in the example above you run remove dupl and obtain nodup files output_pairs_lane1 txt nodupl output_pairs_lane2 txt nodupl output_singles_mates1_lane1 txt nodupl output_singles_mates1_lane2 txt nodupl output_singles_mates2_lane1 txt nodupl output_singles_mates2_lane2 txt nodupl Then you wish to add output_pairs_lane3 txt to pairs_results txt output_pairs_lanel txt output_pairs_lane2 txt output_pairs_lane3 txt and to re run remove dupl on all files Make sure your dele
24. s lt integer gt the number of mismatches in the alignment for 3 mate e Original position 1 lt integer gt original position for 5 mate see definition of original position above e Original position 2 lt integer gt original position for 3 mate see definition of original position above Output format for acgt count Starting with version brat 1 1 17 there are two choices for output format The first choice The number of output files will be double the number of input references two for each reference listed in references_names txt file one file for forward strand and the other for reverse strand In each file there are M lines where M is the size of a corresponding reference in base pairs Each line corresponds to a base of a strand and contains counts for As Cs Gs and Ts at that base for all mapped reads i e there are four integers per line from left to right for As Cs Gs and Ts For the reverse strand the counts of As Cs Gs and Ts are given for the reads that are mapped to the reverse strand but the counts are obtained by aligning the reverse complements of these reads with the forward strand Following is an example to illustrate this point Let a read ACCGTT be mapped to a reverse strand at position i then the corresponding forward strand starting at position i is AACGGT and the counts for the reverse strand at positions i i 5 from this read are incremented for the following nucleotides i A it 1 A i 2
25. t for single reads is a single file with reads whose lengths might be different and whose lengths are greater than or equal to 24 bases The output for pairs is four files two for paired end mapping and two for single end mapping Trimming of paired end reads produces two files with single reads if one mate is shorter than the minimum length allowed and the other s length is correct then the mate with the correct length will be output into a corresponding file with single reads Two files for single reads are necessary because BS mapping for 5 and 3 mates is different Each file contains a single line raw reads format with the following fields e Read lt string gt a read after using trim e Start lt int gt the number of bases trimmed at the beginning of the original read e End lt int gt the number of bases trimmed at the end of the original read 5 DETAILS ON ACGT COUNT Let us denote a mapping of a base in a read to a base in the reference as C C T C AG G gt G Initially we thought that if a read maps to a positive strand then the mappings C C and T C contribute to the count of methylation level of cytocines of the positive strand Similarly if the reverse complement of a read maps to the positive strand equivalently a read maps to the negative strand then the mappings A gt G and G gt G contribute to the count of methylation level of cytocines of the negative strand Let us illustrate this idea in Figures
26. tal number of reads each read is counted i e a pair has 2 reads Then the space required for both programs is bound as follows Spaceprar 269 10 2 4N 24R 3 8 N Bytes Spaceprat targe 269 10 47 24R 3 8 T Bytes Spaceprat targe 269 10 2 47 24R 3 8 T Bytes when option S is provided Whenever there is a space limitation the user can choose to use BRAT large Here is an example of space usage with paired end BS mapping with non BS mismatches mismatches other than T to C and A to G BRAT uses 2 5 GB on 1 million pairs and human chromosome 1 and BRAT large on 10 million pairs and an entire human genome uses 1 7 GB or 2 7GB with option S In this version BRAT allows for non BS mismatches with the restriction on the number of non BS mismatches in the first X bases for BRAT and X bases for BRAT large where X is a threshold specified with the option f X cannot be smaller than 24 or larger than 64 BRAT guarantees to map all reads that could be mapped to the genome with up to one non BS mismatch in the first X bases of reads A user can specify any number of non BS mismatches with the option m and as long as there is only one non BS mismatch in the first X bases of a read BRAT will map this read BRAT large does not allow for non BS mismatches in the first X bases of reads If a user specifies any number of non BS mismatches with the option m BRAT large will map all reads that could be mapped perfectly or with B
27. te existent files with extension nodup rm output_pairs_lane1 txt nodupl rm output_pairs_lane2 txt nodupl rm output_singles_mates1_lane1 txt nodupl rm output_singles_mates1_lane2 txt nodup rm output_singles_mates2_lane1 txt nodupl rm output_singles_mates2_lane2 txt nodupl and only then run remove dupl If you don t remove these files you will have previous output plus new output for example if output_pairs_lane1 txt nodupl had 100 lines then re running remove dupl without removing this file will result in 200 lines A command to run acgt count To count mapped As Cs Gs and Ts at each base of forward and reverse strands of the references use acgt count acgt count r references_names txt P prefix p pairs_results txt s single_results txt the file references_names txt contains the names of FASTA files with the references which are needed to calculate the sizes of the references The output will be in two files per a reference prefix_forw_aReference_name and prefix_rev_aReference_name The option p is to specify the results of paired end mapping if any and s is to specify the results of single end mapping if any NOTE the file pairs_results txt does not contain the actual results it contains the names of the files with the results for paired end mapping and similarly single_results txt file contains the names of the files with the actual results for single end mapping At least one of these options must be provi
28. tion P differs only by not specifying option r If option bs was passed to brat large build then this option must be passed to brat large In other words for mapping normal and bisulfite treated reads two different hashing indices must be built The same true when option S is used if a user wishes to use this option and P option then S must be used with brat large build When P is used during mapping option f will have the same value that was set with brat large build If a user does not specify this option the default value 24 will be used If a user wishes to speed up mapping time and to use option P then the user could set option f to a higher value with brat large build General rule for using option P if a user wishes to change either of the parameters f S or bs the user must apply the same parameters with brat large build Default options for brat large build are f 24 S is not set i e preference is given to slower mapping but also smaller space usage and normal mapping i e bs is not set Commands Options A specifies 3 mates in our examples above either of prefix_reads2 txt or prefix_mates2 txt files must be used with this option If the user does not specify this option and provides either of the files prefix_mates2 txt or prefix_reads2 txt as input reads for single end mapping the mapping will NOT be correct u is used when a user wishes to output unmapped reads The output will be in prefix_reads1 txt unm file
29. y maximum insert size for paired end mapping the maximum distance allowed between the leftmost ends of the mapped mates on forward strand default is 300 0 lt string gt to specify the file with the results of mapping M is used when a user wishes to output ambiguous reads The output will be in prefix_reads1 txt amb for single reads and in prefix_reads1 txt amb and prefix_reads2 txt amb files with paired end reads a single read per line This option does not output the mapping locations for ambiguous reads but just the reads themselves m lt integer gt the maximum number of non BS mismatches allowed by a user default is 0 f lt integer gt the number of the first bases of a read where the restriction on the number of non BS mismatches applies for BRAT only one non BS mismatch is allowed in the first lt integer gt bases and for BRAT large NO non BS mismatches are allowed in the first lt integer gt bases for BRAT default is 36 and for BRAT large default is 24 S to specify speed mode for BRAT large Space usage with this option doubles but running time is about three times faster P lt directory name gt To use this option that allows to separate hashing of the genome from mapping of reads L lt integer gt the smallest value of the range of base quality scores in ASCII representation default is 33 The table below gives examples of different quality scores and their range in ASCII representation from Wikipedia Th
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