D00360:96:H2YLYBCXX:1:2110:18364:84053    353    seq1_len154_cov5    1    1    92S59M8I17M1D6M1D67M    seq30532_len2134_cov76    1    0    AAAAAAAAAAAAAAAAAAAAAAAACCCTGTCTCTAATAAAATACAAACAATTAGCCGGGCATGGTGGCACGCGCCTTTAGTCCCAGCTACTAGGGAGGCTGAGGCAGGGGAATTGTTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCGGAGTTTTTTTCACTGCACTCCAGCCTGGTGACAAATCAAAAATCCATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAACAACAAA

DDDDDHIIIIIIII<EHHII?EHH0001111111<11<DEH1D1<FH1D<<<C<@GEHD</<11<101<1D<<C<E0D11<<1<D?1F1CC1DE110C0D1011100////0DD<1=@1=FGHCDHH<FG<D0<<<EF?CE<00<<0<D//0;<:D/;///;8F.;/.8.8......88.9........-8BBGADHIIHD?>D?HH<,>=HHDD,5CHDCDHD><,,,--8----8-8--    NM:i:25    MD:Z:16A21C16C0A3T15^A6^G1G0T0G3C2T0G1C41A4A2A3    AS:i:49    XS:i:42    SA:Z:seq13646_len513_cov63,125,-,103S125M21S,1,11;    RG:Z:chr22

>seq1_len154_cov5
GGGAGGCTGAGGCAGGAGAATTGTTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGCACTCCAGCCTGGATGACAAGAGTGAAACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

>>> bam_file = pysam.AlignmentFile(infile, "rb")
>>> for line in bam_file:
...     if 300 < line.query_alignment_length < 500:
...             break
...
>>> line.query_alignment_length
321
>>> line.seq
'CTCTTCATCACGTCACTTGTCCATGTCAATGGCTACAGGTTGGAAGTTTGGCCGCGGAGGGTGGGCAGGCAAGAGAAAGAAATCAGATAGGGCAGATGGTAGGGTAAAAGGAGGGGGTTAAGTGCAAATTGTCTACTGTTTGCAAATGGGAAGCATGTGATTGTTAAAATTTATACGATAAACCTTCTCATCATGTTGAGTCTCATGCTTGCGCCAAGAAGATCGGGTTCGGCGGGTCAAGCTGATAAGCAACTTGGGCAGCAAAGTCGTTCAGTGATACAAAATCATGTGCAAAAATCACAAGCATTCTTATAAACACCA'
>>> line.cigarstring
'5148H3M1I2M1I4M2I3M3I3M1D2M1I4M3I1M1I4M1D10M1D5M4I4M3D1M3I18M1I1M1I1M1I4M4D2M1I2M2I4M2I4M1I4M5I4M1I9M1I14M3I1M1I2M2I7M2I7M3I2M1I5M2I1M1I2M1I1M1I4M5I3M1I2M3I1M1I4M4I3M4I2M2I1M3I19M3I10M2I4M1I11M1D27M2I6M25212H'
>>> line.reference_name
'Backbone_1'
>>> line.reference_start
7164
>>> line.reference_end
7413
>>> line.get_tag('NM')
100

>>> ref = 'CTCTCTCACCACTCCTATTCAACACAGTGTTGGAAGTTCTGGACAGGGCAATCAGGCAAGAGAAAGAAATAAAGGGTATTCAATTAGGAAAAGAGGAAGTCAAATTGTCCCCGTTTGCAGATGACATGATTGTATATTTAGAAAACCCCACTGTTTCAGCCCCAAATCTCCTTAAGCTGATAAGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATGTGCAAAAATCACAAGCATTCTTATACACCA'

def formatSeqByCigar(seq, cigar):'''Input: query_alignment_sequence and cigar from sam fileOutput: formatSeqPurpose: make sure the pos is one to one correspondence(seq to ref)'''formatSeq = ''pointer = 0; qstart = 0; qend = -1; origin_seq_len = 0if cigar[0][0] == 4 or cigar[0][0] == 5:qstart = cigar[0][1]if cigar[-1][0] == 4 or cigar[-1][0] == 5:qend = - cigar[-1][1] - 1  # fushu countfor pair in cigar:operation = pair[0]cigar_len = pair[1]if operation == 0: # 0==MformatSeq += seq[pointer:(pointer+cigar_len)]pointer += cigar_lenorigin_seq_len += cigar_lenelif operation == 1: # 1==Ipointer += cigar_lenorigin_seq_len += cigar_lenelif operation == 2: # 2==DformatSeq += 'D'*cigar_lenelif operation == 4 or operation == 5: # 5==Horigin_seq_len += cigar_lencontinueelse:print (cigar)raise TypeError("There are cigar besides S/M/D/I/H\n")return formatSeq, qstart, qend, origin_seq_len

>>> i = 0
>>> count = 0
>>> while i < len(ref):if formatSeq[i] != ref[i]:count += 1i += 1>>> count
17
>>> formatSeq.count('D')
11

>>> cigarstring = '5148H3M1I2M1I4M2I3M3I3M1D2M1I4M3I1M1I4M1D10M1D5M4I4M3D1M3I18M1I1M1I1M1I4M4D2M1I2M2I4M2I4M1I4M5I4M1I9M1I14M3I1M1I2M2I7M2I7M3I2M1I5M2I1M1I2M1I1M1I4M5I3M1I2M3I1M1I4M4I3M4I2M2I1M3I19M3I10M2I4M1I11M1D27M2I6M25212H'
>>> cigarstring.count('I')
41
>>> cigarstring.count('D')
6