BWA程序調試記錄

• 1 wgsim生成read文件

./wgsim -N100000 -1100 -d0 -S11 -e0 -r10 xx.fa xx.fq /dev/null

• 2 Index生成庫文件

在Program arguments中輸入 index xx.fa，運行後生成5個文件

pac：2位編碼後的文件

ann、amb：臨時文件

bwt：包括BWT數組和OCC信息

sa：由bwt文件生成的後綴數組

• 3 bwa mem 比對

bwa mem -R '@RG\tID:foo\tSM:bar\tLB:library1' <ref.fa> <read1.fa> <read2.fa>  >  lane.sam

例子：

bwa mem -t 12 -o test.sam 15.fa 15.fq

        參數詳解：

-o FILE-   sam file to output results to [stdout]

-t INT	Number of threads [1]  比對線程數目，每一個線程默認讀取1千萬bp
-k INT	Minimum seed length. Matches shorter than INT will be missed. The alignment speed is usually insensitive to this value unless it significantly deviates 20. [19]
-w INT	Band width. Essentially, gaps longer than INT will not be found. Note that the maximum gap length is also affected by the scoring matrix and the hit length, not solely determined by this option. [100]
-d INT	Off-diagonal X-dropoff (Z-dropoff). Stop extension when the difference between the best and the current extension score is above |i-j|*A+INT, where i and j are the current positions of the query and reference, respectively, and A is the matching score. Z-dropoff is similar to BLAST’s X-dropoff except that it doesn’t penalize gaps in one of the sequences in the alignment. Z-dropoff not only avoids unnecessary extension, but also reduces poor alignments inside a long good alignment. [100]
-r FLOAT	Trigger re-seeding for a MEM longer than minSeedLen*FLOAT. This is a key heuristic parameter for tuning the performance. Larger value yields fewer seeds, which leads to faster alignment speed but lower accuracy. [1.5]
-c INT	Discard a MEM if it has more than INT occurence in the genome. This is an insensitive parameter. [10000]
-P	In the paired-end mode, perform SW to rescue missing hits only but do not try to find hits that fit a proper pair. 雙末端序列，運行 SW 模式，進一步比對未成功匹配的reads，成功匹配的reads保留原樣。
-A INT	Matching score. [1]
-B INT	Mismatch penalty. The sequence error rate is approximately: {.75 * exp[-log(4) * B/A]}. [4]
-O INT	Gap open penalty. [6]
-E INT	Gap extension penalty. A gap of length k costs O + k*E (i.e. -O is for opening a zero-length gap). [1]
-L INT	Clipping penalty. When performing SW extension, BWA-MEM keeps track of the best score reaching the end of query. If this score is larger than the best SW score minus the clipping penalty, clipping will not be applied. Note that in this case, the SAM AS tag reports the best SW score; clipping penalty is not deducted. [5]
-U INT	Penalty for an unpaired read pair. BWA-MEM scores an unpaired read pair as scoreRead1+scoreRead2-INT and scores a paired as scoreRead1+scoreRead2-insertPenalty. It compares these two scores to determine whether we should force pairing. [9]
-p	Assume the first input query file is interleaved paired-end FASTA/Q. See the command description for details.
-R STR	Complete read group header line. ’\t’ can be used in STR and will be converted to a TAB in the output SAM. The read group ID will be attached to every read in the output. An example is ’@RG\tID:foo\tSM:bar’. [null]
-T INT	Don’t output alignment with score lower than INT. This option only affects output. [30]
-a	Output all found alignments for single-end or unpaired paired-end reads. These alignments will be flagged as secondary alignments.
-C	Append append FASTA/Q comment to SAM output. This option can be used to transfer read meta information (e.g. barcode) to the SAM output. Note that the FASTA/Q comment (the string after a space in the header line) must conform the SAM spec (e.g. BC:Z:CGTAC). Malformated comments lead to incorrect SAM output.
-H	Use hard clipping ’H’ in the SAM output. This option may dramatically reduce the redundancy of output when mapping long contig or BAC sequences.
-M	Mark shorter split hits as secondary (for Picard compatibility). 將較短的 split hits 標記爲secondary；用於兼容picard
-v INT	Control the verbose level of the output. This option has not been fully supported throughout BWA. Ideally, a value 0 for disabling all the output to stderr; 1 for outputting errors only; 2 for warnings and errors; 3 for all normal messages; 4 or higher for debugging. When this option takes value 4, the output is not SAM. [3]