comparison mzsqlite_psm_align.py @ 0:492f98d89e26 draft

planemo upload for repository https://github.com/jj-umn/galaxytools/tree/master/mzsqlite_psm_align commit 88e2fb9c31fbd687a0956924a870137d1fb9bee3-dirty

0:492f98d89e26

#!/usr/bin/env python
"""
#
#------------------------------------------------------------------------------
#                         University of Minnesota
#         Copyright 2017, Regents of the University of Minnesota
#------------------------------------------------------------------------------
# Author:
#
#  James E Johnson
#
#------------------------------------------------------------------------------
"""

from __future__ import print_function

import argparse
import re
import sys
import sqlite3 as sqlite
from time import time


from Bio.Seq import reverse_complement, translate

## from bedutil import bed_from_line

## import digest

## from ensembl_rest import get_cdna

import pysam
from twobitreader import TwoBitFile

from  profmt import PROBAM_DEFAULTS,ProBAM,ProBAMEntry,ProBED,ProBEDEntry

"""
inputs 
  proBed 
  mzIdentML 
  twobit
  bam

inputs 
  mz.sqlite
  genomic.mapping 
  bam

CREATE TABLE spectrum_identification_results (id TEXT PRIMARY KEY, spectraData_ref TEXT, spectrumID TEXT, spectrumTitle TEXT);
CREATE TABLE spectrum_identification_result_items (id TEXT PRIMARY KEY, spectrum_identification_result_ref TEXT, passThreshold TEXT, rank INTEGER, peptide_ref TEXT, calculatedMassToCharge FLOAT, experimentalMassToCharge FLOAT, chargeState INTEGER);
CREATE TABLE peptide_evidence (id TEXT PRIMARY KEY, dBSequence_ref TEXT, isDecoy TEXT, pre TEXT, post TEXT, start INTEGER, end INTEGER, peptide_ref TEXT);
CREATE TABLE db_sequence (id TEXT PRIMARY KEY , accession TEXT, searchDatabase_ref TEXT, description TEXT, sequence TEXT, length INTEGER);

SELECT 
FROM spectrum_identification_result_items siri
 JOIN peptide_evidence pe ON siri.peptide_ref = pe.peptide_ref
 JOIN db_sequence dbs ON pe.dBSequence_ref = 
WHERE pe.isDecoy = 'false'

SELECT 
  psm.spectrumID,
  psm.spectrumTitle as "QNAME",
  psm.id,
  psm.sequence,
  psm.passThreshold,
  psm."PeptideShaker PSM confidence",
  psm."PeptideShaker PSM score",
  pe.start,
  pe.end,
  pe.pre,
  pe.post,
  pe.dBSequence_ref
FROM psm_entries psm 
JOIN peptide_evidence pe ON psm.id = pe.peptide_ref
JOIN db_sequence dbs ON pe.dBSequence_ref = dbs.accession
WHERE pe.isDecoy = 'false'
AND pe.peptide_ref = 'SFYPEEVSSMVITK_15.99491461956-ATAA-10'
ORDER BY psm.spectrumID
  

proBed to SQLite
or index proBed

for psm in psms:
  beds = get_bed(protein_acc)
  cds = ''
  for bed in beds:
    bed.seq = twobit[bed.chrom][bed.start,bed.end]
    cds += bed.get_cds()
  refprot = translate(cds)

def read_bed(path):
    pdict = dict()
    prog = re.compile('^([^\t]+\t[^\t]+\t[^\t]+\t([^\t]+)\t.*)$')
    with open(path,'r') as bed:
        for i,line in enumerate(bed):
            m = prog.match(line)
            prot = m.groups()[1]
            pdict[prot] = m.groups()[0]
    return pdict
  
from pyteomics import mzid
    with mzid.reader(args.mzid) as mzidrdr:
        for psm in mzidrdr:
            SpectrumIdentificationItems = psm['SpectrumIdentificationItem']
            for SpectrumIdentificationItem in SpectrumIdentificationItems:
                PeptideEvidenceRef = SpectrumIdentificationItem['PeptideEvidenceRef']
                PepEvs = [r['peptideEvidence_ref'] for r in PeptideEvidenceRef]
                for PepEv in PepEvs:
                    PepRef = mzidrdr[PepEv]
                    dBSequence_ref = PepRef['dBSequence_ref']

spectrum_peptides =  count(distinct sequence) FROM psm_entries WHERE 

1 QNAME String Query template NAME Spectrum name *                                psm.spectrumTitle
2 FLAG Int Bitwise FLAG Bitwise FLAG                                              map.strand
3 RNAME String Reference sequence NAME Reference sequence NAME *                  map.chrom
4 POS Int 1-based leftmost mapping POSition 1-based leftmost mapping POSition     map.start
5 -MAPQ Int MAPping Quality (Phred-scaled) - 255
6 CIGAR String Extended CIGAR string (operations: MIDN) CIGAR string *            map.cigar
7 -RNEXT String Mate Reference NAME ('=' if same as RNAME) - *
8 -PNEXT Int 1-Based leftmost Mate POSition - 0
9 TLEN Int observed Template LENgth - 0                                           
10 SEQ String segment SEQuence Coding sequence *                                  genomic.seq
11 -QUAL String Query QUALity (ASCII-33=Phred base quality) - *                   

1 QNAME    psm.spectrumTitle
2 FLAG     map.strand
3 RNAME    map.chrom
4 POS      map.start
5 -MAPQ 
6 CIGAR    map.cigar
7 -RNEXT 
8 -PNEXT 
9 -TLEN                
10 SEQ     genomic.seq
11 -QUAL              
                                                                                  
'NH' : 'i' genomic_locations 
'XO' : 'Z' 
'XL' : 'i' spectrum_peptides 
'XP' : 'Z' psm.sequence 
'YP' : 'Z' peptide_evidence.dBSequence_ref
'XF' : 'Z' reading_frame
'XI' : 'f' 
'XB' : 'Z' 
'XR' : 'Z' 
'YB' : 'Z' 
'YA' : 'Z' 
'XS' : 'f' 
'XQ' : 'f' 
'XC' : 'i' 
'XA' : 'i' 
'XM' : 'Z' 
'XN' : 'i' 
'XT' : 'i' 
'XE' : 'i' 
'XG' : 'A' 
'XU' : 'Z' 

'NH' : 'i', #number of genomic locations to which the peptide sequence maps
'XO' : 'Z', #uniqueness of the peptide mapping
'XL' : 'i', #number of peptides to which the spectrum maps
'XP' : 'Z', #peptide sequence
'YP' : 'Z', #Protein accession ID from the original search result
'XF' : 'Z', #Reading frame of the peptide (0, 1, 2)
'XI' : 'f', #Peptide intensity
'XB' : 'Z', #massdiff; experimental mass; calculated mass massdiff can be calculated by experimental mass - calculated mass. If any number is unavailable, the value should be left blank (such as 0.01;;).
'XR' : 'Z', #reference peptide sequence
'YB' : 'Z', #Preceding amino acids (2 AA, B stands for before).
'YA' : 'Z', #Following amino acids (2 AA, A stands for after).
'XS' : 'f', #PSM score
'XQ' : 'f', #PSM FDR (i.e. q-value or 1-PEP).
'XC' : 'i', #peptide charge
'XA' : 'i', #Whether the peptide is annotated 0:yes; 1:parially unknown; 2:totally unknown;
'XM' : 'Z', #Modifications
'XN' : 'i', #Number of missed cleavages in the peptide (XP)
'XT' : 'i', #Enzyme specificity
'XE' : 'i', #Enzyme used in the experiment
'XG' : 'A', #Peptide type
'XU' : 'Z', #URI


Datatype Field name Description Origin
RNAME	string          chrom                     map.chrom      Reference sequence chromosome 
POS	uint            chromStart                map            Start position of the first DNA base 
	uint            chromEnd                  map            End position of the last DNA base 
QNAME	string          name                      spectrum.title     Unique name 
	uint            score                           Score 
	char[1]         strand                          + or - for strand 
	uint            thickStart                      Coding region start 
	uint            thickEnd                        Coding region end 
	uint            reserved                        Always 0 
	int             blockCount                      Number of blocks 
	int[blockCount] blockSizes                      Block sizes 
	int[blockCount] chromStarts                     Block starts 
YP	string          proteinAccession                Protein accession number 
XP	string          peptideSequence                 Peptide sequence 
XO	string          uniqueness                      Peptide uniqueness 
	string          genomeReferenceVersion          Genome reference version number 
XS	double          psmScore                        PSM score 
XQ	double          fdr                             Estimated global false discovery rate 
XM	string          modifications                   Post-translational modifications 
XC	int             charge                          Charge value 
XB	double          expMassToCharge                 Experimental mass to charge value 
XB	double          calcMassToCharge                Calculated mass to charge value 
int             psmRank                         Peptide-Spectrum Match rank. 
string          datasetID                       Dataset Identifier 
string          uri                             Uniform Resource Identifier 

XG
    N  Normal peptide. The peptide sequence is contained in the reference protein sequence.
    V  Variant peptide. A single amino acid variation (SAV) is present as compared to the reference.
    W  Indel peptide. An insertion or deletion is present as compared to the reference.
    J  Novel junction peptide. A peptide that spans a novel exon-intron boundary as compared to the reference.
    A  Alternative junction peptide. A peptide that spans a non-canonical exon-intron boundary as compared to the reference.
    M  Novel exon peptide. A peptide that resides in a novel exon that is not present in the reference.
    C  Cross junction peptide. A peptide that spans through a splice site (partly exonic - partly intronic).
    E  Extension peptide. A peptide that points to a non-canonical N-terminal protein extension.
    B  3' UTR peptide. A peptide that maps to the 3' UTR region from the reference.
    O  Out-of-frame peptide. A peptide that is translated from an alternative frame as compared to the reference.
    T  Truncation peptide. A peptide that points to a non-canonical N-terminal protein truncation.
    R  Reverse strand peptide. A peptide that is derived from translation of the reverse strand of the reference.
    I  Intron peptide. A peptide that is located in an intronic region of the reference isoform.
    G  Gene fusion peptide. An (onco-) peptide that spans two exons of different genes, through gene-fusion.
    D  Decoy peptide. A peptide that maps to a decoy sequence from the MS-based search strategy.
    U  Unmapped peptide. A peptide that could not be mapped to a reference sequence.
    X  Unknown.



SELECT distinct chrom, CASE WHEN strand = '+' THEN start + cds_offset - cds_start ELSE end - cds_offset - cds_start END as "pos"
FROM feature_cds_map
WHERE name = acc_name AND cds_offset >= cds_start AND cds_offset < cds_end

sqlite> select * from feature_cds_map WHERE name = 'pre_STRG.28813.4_j_5350_5470';
pre_STRG.28813.4_j_5350_5470|chr7|5074750|5074857|+|0|107
pre_STRG.28813.4_j_5350_5470|chr7|5075140|5075153|+|107|120



SELECT 
  pe.isDecoy,
  pe.dBSequence_ref,
  pe.start,
  pe.end,
  sr.spectrumTitle,
  si.rank,
  si.chargeState,
  si.calculatedMassToCharge,
  si.experimentalMassToCharge
FROM spectrum_identification_results sr 
JOIN spectrum_identification_result_items si ON si.spectrum_identification_result_ref = sr.id
JOIN peptide_evidence pe ON si.peptide_ref = pe.peptide_ref
WHERE si.id = 'SII_7389_1' 
ORDER BY si.rank;

SELECT pe.isDecoy, pe.dBSequence_ref, pe.start, pe.end, sr.spectrumTitle, si.rank, si.chargeState, si.calculatedMassToCharge, si.experimentalMassToCharge
FROM spectrum_identification_results sr 
JOIN spectrum_identification_result_items si ON si.spectrum_identification_result_ref = sr.id
JOIN peptide_evidence pe ON si.peptide_ref = pe.peptide_ref
WHERE si.id = 'SII_7389_1' 
ORDER BY si.rank;



CREATE TABLE spectrum_identification_results (id TEXT PRIMARY KEY, spectraData_ref TEXT, spectrumID TEXT, spectrumTitle TEXT);
CREATE TABLE spectrum_identification_result_items (id TEXT PRIMARY KEY, spectrum_identification_result_ref TEXT, passThreshold TEXT, rank INTEGER, peptide_ref TEXT, calculatedMassToCharge FLOAT, experimentalMassToCharge FLOAT, chargeState INTEGER);
CREATE TABLE peptide_evidence (id TEXT PRIMARY KEY, dBSequence_ref TEXT, isDecoy TEXT, pre TEXT, post TEXT, start INTEGER, end INTEGER, peptide_ref TEXT);
CREATE TABLE db_sequence (id TEXT PRIMARY KEY , accession TEXT, searchDatabase_ref TEXT, description TEXT, sequence TEXT, length INTEGER);

{'write_probed': 0.08575654029846191, 'PSM_QUERY': 4.704349040985107, 'get_cds': 0.21015286445617676, 'SPECTRUM_PEPTIDES_QUERY': 32.92655086517334, 'PEPTIDE_ACC_QUERY': 425.11919951438904, 'get_mapping': 1.5911591053009033, 'GENOMIC_POS_QUERY': 10.909647226333618}
"""



def regex_match(expr, item):
    return re.match(expr, item) is not None


def regex_search(expr, item):
    return re.search(expr, item) is not None


def regex_sub(expr, replace, item):
    return re.sub(expr, replace, item)


def get_connection(sqlitedb_path, addfunctions=True):
    conn = sqlite.connect(sqlitedb_path)
    if addfunctions:
        conn.create_function("re_match", 2, regex_match)
        conn.create_function("re_search", 2, regex_search)
        conn.create_function("re_sub", 3, regex_sub)
    return conn

PSM_QUERY = """\
SELECT 
  pe.dBSequence_ref,
  pe.start,
  pe.end,
  pe.pre,
  pe.post,
  pep.sequence,
  sr.id,
  sr.spectrumTitle,
  si.rank,
  si.chargeState,
  si.calculatedMassToCharge,
  si.experimentalMassToCharge,
  si.peptide_ref
FROM spectrum_identification_results sr 
JOIN spectrum_identification_result_items si ON si.spectrum_identification_result_ref = sr.id
JOIN peptide_evidence pe ON si.peptide_ref = pe.peptide_ref
JOIN peptides pep ON pe.peptide_ref = pep.id
WHERE pe.isDecoy = 'false'
ORDER BY sr.spectrumTitle,si.rank
"""

PEP_MODS_QUERY = """\
SELECT location, residue, name, modType, '' as "unimod"
FROM peptide_modifications
WHERE peptide_ref = :peptide_ref
ORDER BY location, modType, name
"""

#number of peptides to which the spectrum maps
## spectrum_identification_results => spectrum_identification_result_items -> peptide_evidence 
SPECTRUM_PEPTIDES_QUERY = """\
SELECT count(distinct pep.sequence)
FROM spectrum_identification_results sr
JOIN spectrum_identification_result_items si ON si.spectrum_identification_result_ref = sr.id
JOIN peptide_evidence pe ON si.peptide_ref = pe.peptide_ref
JOIN peptides pep ON pe.peptide_ref = pep.id
WHERE pe.isDecoy = 'false'
AND sr.id = :sr_id
GROUP BY sr.id
"""
#number of genomic locations to which the peptide sequence maps
#uniqueness of the peptide mapping
## peptides => peptide_evidence -> db_sequence -> location
## proteins_by_peptide
PEPTIDE_ACC_QUERY = """\
SELECT 
  pe.dBSequence_ref,
  pe.start,
  pe.end
FROM peptide_evidence pe
JOIN peptides pep ON pe.peptide_ref = pep.id
WHERE pe.isDecoy = 'false'
AND pep.sequence = :sequence
"""

MAP_QUERY = """\
SELECT distinct * 
FROM feature_cds_map 
WHERE name = :acc 
AND :p_start < cds_end 
AND :p_end >= cds_start 
ORDER BY name,cds_start,cds_end
"""

GENOMIC_POS_QUERY = """\
SELECT distinct chrom, CASE WHEN strand = '+' THEN start + :cds_offset - cds_start ELSE end - :cds_offset - cds_start END as "pos"
FROM feature_cds_map
WHERE name = :acc 
AND :cds_offset >= cds_start 
AND :cds_offset < cds_end
"""

FEATURE_CONTAIN_QUERY = """\
SELECT id,seqid,start,end,featuretype,strand,frame
FROM features
WHERE seqid = :seqid AND start <= :start AND end >= :end 
AND strand = :strand AND featuretype = :ftype
"""

FEATURE_OVERLAP_QUERY = """\
SELECT id,seqid,start,end,featuretype,strand,frame
FROM features
WHERE seqid = :seqid 
AND :end >= start AND :start <= end 
AND strand = :strand AND featuretype = :ftype
"""

FEATURE_ANY_QUERY = """\
SELECT id,seqid,start,end,featuretype,strand,CAST(frame AS INTEGER) as "frame", CAST(frame AS INTEGER)==:frame as "in_frame"
FROM features
WHERE seqid = :seqid 
AND :end >= start AND :start <= end 
AND featuretype in ('CDS','five_prime_utr','three_prime_utr','transcript')
ORDER BY strand == :strand DESC, featuretype, 
start <= :start AND end >= :end DESC,
in_frame DESC, end - start, start DESC, end
"""

def __main__():
    parser = argparse.ArgumentParser(
        description='Generate proBED and proBAM from mz.sqlite')
    parser.add_argument('mzsqlite', help="mz.sqlite converted from mzIdentML")
    parser.add_argument('genomic_mapping_sqlite', help="genomic_mapping.sqlite with feature_cds_map table")
    parser.add_argument(
        '-R', '--genomeReference', default='Unknown',
        help='Genome reference sequence in 2bit format')
    parser.add_argument(
        '-t', '--twobit', default=None,
        help='Genome reference sequence in 2bit format')
    parser.add_argument(
        '-r', '--reads_bam', default=None,
        help='reads alignment bam path')
    parser.add_argument(
        '-g', '--gffutils_file', default=None,
        help='gffutils GTF sqlite DB')
    parser.add_argument(
        '-B', '--probed', default=None,
        help='proBed path')
    parser.add_argument(
        '-s', '--prosam', default=None,
        help='proSAM path')
    parser.add_argument(
        '-b', '--probam', default=None,
        help='proBAM path')
    parser.add_argument(
        '-l', '--limit', type=int, default=None,
        help='limit numbers of PSMs for testing')
    parser.add_argument('-v', '--verbose', action='store_true', help='Verbose')
    parser.add_argument('-d', '--debug', action='store_true', help='Debug')
    args = parser.parse_args()

    def get_sequence(chrom, start, end):
        if twobit:
            if chrom in twobit and 0 <= start < end < len(twobit[chrom]):
                return twobit[chrom][start:end]
            contig = chrom[3:] if chrom.startswith('chr') else 'chr%s' % chrom
            if contig in twobit and 0 <= start < end < len(twobit[contig]):
                return twobit[contig][start:end]
            return ''
        return None

    twobit = TwoBitFile(args.twobit) if args.twobit else None
    samfile = pysam.AlignmentFile(args.reads_bam, "rb" ) if args.reads_bam else None
    seqlens = twobit.sequence_sizes()

    probed = open(args.probed,'w') if args.probed else sys.stdout
    
    gff_cursor = get_connection(args.gffutils_file).cursor() if args.gffutils_file else None
    map_cursor = get_connection(args.genomic_mapping_sqlite).cursor()
    mz_cursor = get_connection(args.mzsqlite_file).cursor()

    unmapped_accs = set()
    timings = dict()
    def add_time(name,elapsed):
        if name in timings:
            timings[name] += elapsed
        else:
            timings[name] = elapsed

    XG_TYPES = ['N','V','W','J','A','M','C','E','B','O','T','R','I','G','D','U','X','*']
    FT_TYPES = ['CDS','five_prime_utr','three_prime_utr','transcript']
    def get_peptide_type(exons):
        ## XG classify peptide
        ##     N  Normal peptide. The peptide sequence is contained in the reference protein sequence.
        ##     V  Variant peptide. A single amino acid variation (SAV) is present as compared to the reference.
        ##     W  Indel peptide. An insertion or deletion is present as compared to the reference.
        ##     J  Novel junction peptide. A peptide that spans a novel exon-intron boundary as compared to the reference.
        ##     A  Alternative junction peptide. A peptide that spans a non-canonical exon-intron boundary as compared to the reference.
        ##     M  Novel exon peptide. A peptide that resides in a novel exon that is not present in the reference.
        ##     C  Cross junction peptide. A peptide that spans through a splice site (partly exonic - partly intronic).
        ##     E  Extension peptide. A peptide that points to a non-canonical N-terminal protein extension.
        ##     B  3' UTR peptide. A peptide that maps to the 3' UTR region from the reference.
        ##     O  Out-of-frame peptide. A peptide that is translated from an alternative frame as compared to the reference.
        ##     T  Truncation peptide. A peptide that points to a non-canonical N-terminal protein truncation.
        ##     R  Reverse strand peptide. A peptide that is derived from translation of the reverse strand of the reference.
        ##     I  Intron peptide. A peptide that is located in an intronic region of the reference isoform.
        ##     G  Gene fusion peptide. An (onco-) peptide that spans two exons of different genes, through gene-fusion.
        ##     D  Decoy peptide. A peptide that maps to a decoy sequence from the MS-based search strategy.
        ##     U  Unmapped peptide. A peptide that could not be mapped to a reference sequence.
        ##     X  Unknown.

        peptide_type = '*'
        if gff_cursor:
            ts = time()
            etypes = ['*'] * len(exons)
            efeatures = [None] * len(exons)
            if args.debug:
                print('exons:%d\t%s'% (len(exons),etypes),file=sys.stderr)
            for i,exon in enumerate(exons):
                (acc,gc,gs,ge,st,cs,ce) = exon
                fr = cs % 3
                if args.debug:
                    print('exon:\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s' % (acc,gc,gs,ge,st,cs,ce,fr),file=sys.stderr)
                ft_params = {"seqid" : str(gc).replace('chr',''), "start" : gs, "end" : ge, 'strand' : st, 'frame' : fr, 'ftype' : 'CDS'}
                features = [f for f in gff_cursor.execute(FEATURE_ANY_QUERY,ft_params)]
                efeatures[i] = features
            for i,exon in enumerate(exons):
                (acc,gc,gs,ge,st,cs,ce) = exon
                for f in efeatures[i]:
                    (id,seqid,start,end,featuretype,strand,frame,in_frame) = f
                    if args.debug:
                        print('feat:\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s' % (id,seqid,start,end,featuretype,strand,frame,in_frame),file=sys.stderr)
                    if strand == st:
                        if start <= gs and ge <= end:
                            if in_frame:
                               etypes[i] = 'N'
                               break
                            elif XG_TYPES.index('O') < XG_TYPES.index(etypes[i]):
                               etypes[i] = 'O'
                        break
                    else:
                        if XG_TYPES.index('O') < XG_TYPES.index(etypes[i]):
                            etypes[i] = 'O'
                peptide_type = etypes[i]
            te = time()   
            add_time('pep_type',te - ts)
        return peptide_type
    def classify_exon(exon,exons,features):
            ##     N  Normal peptide. The peptide sequence is contained in the reference protein sequence.
            # 1 exon, contained, in_frame
            # 2+ exons, contained, in_frame, on_exon_boundary
            ##     V  Variant peptide. A single amino acid variation (SAV) is present as compared to the reference.
            # 1 exon, contained, in_frame, AA_mismatch
            # 2+ exons, contained, in_frame, on_exon_boundary, AA_mismatch
            ##     W  Indel peptide. An insertion or deletion is present as compared to the reference.
            # 1 exon, contained, in_frame, AA_mismatch
            # 2+ exons, contained, in_frame, on_exon_boundary or off by 3, AA_mismatch
            ##     J  Novel junction peptide. A peptide that spans a novel exon-intron boundary as compared to the reference.
            # 2+ exons, contained, on_exon_boundary, same transcript, non adjacent exons
            ##     A  Alternative junction peptide. A peptide that spans a non-canonical exon-intron boundary as compared to the reference.
            # 2+ exons, contained, on_exon_boundary, same transcript, non adjacent exons
            ##     M  Novel exon peptide. A peptide that resides in a novel exon that is not present in the reference.
            ##     C  Cross junction peptide. A peptide that spans through a splice site (partly exonic - partly intronic).
            # 1 exon overlaps but not contained 
            ##     E  Extension peptide. A peptide that points to a non-canonical N-terminal protein extension.
            ##     B  3' UTR peptide. A peptide that maps to the 3' UTR region from the reference.
            # exon overlaps a three_prime_utr
            ##     O  Out-of-frame peptide. A peptide that is translated from an alternative frame as compared to the reference.
            # exon contained but not in_frame
            ##     T  Truncation peptide. A peptide that points to a non-canonical N-terminal protein truncation.
            ##     R  Reverse strand peptide. A peptide that is derived from translation of the reverse strand of the reference.
            ##     I  Intron peptide. A peptide that is located in an intronic region of the reference isoform.
            # exon contained in transcript, not not overlapping any exon
            ##     G  Gene fusion peptide. An (onco-) peptide that spans two exons of different genes, through gene-fusion.
            # exonis from different seqs, strand, or transcripts
            ##     D  Decoy peptide. A peptide that maps to a decoy sequence from the MS-based search strategy.
            ##     U  Unmapped peptide. A peptide that could not be mapped to a reference sequence.
            ##     X  Unknown.
        return '*'

    def get_variant_cds(exons,ref_prot,peptide,pep_cds):
        if ref_prot != peptide and samfile:
            try:
                if args.debug:
                    print('name: %s \nref: %s\npep: %s\n' % (scan_name,ref_prot,peptide), file=sys.stderr)
                ts = time()
                for exon in exons:
                   (acc,chrom,start,end,strand,c_start,c_end) = exon
                   a_start = c_start / 3 * 3
                   a_end = c_end / 3 * 3
                   if ref_prot[a_start:a_end] != peptide[a_start:a_end]:
                       pileup = get_exon_pileup(chrom,start,end)
                       for i, (bi,ai,ao) in enumerate([(i,i / 3, i % 3) for i in range(c_start, c_end)]):
                           if ao == 0 or i == 0:
                               if ref_prot[ai] != peptide[ai]:
                                   codon = get_pep_codon(pileup, bi - c_start, peptide[ai], ao)
                                   if args.debug:
                                       print('%d %d %d   %s :  %s %s %s' % (bi,ai,ao,  peptide[ai], str(pep_cds[:bi]), str(codon), str(pep_cds[bi+3:])), file=sys.stderr)
                                   if codon:
                                       pep_cds = pep_cds[:bi] + codon + pep_cds[bi+3:]
                te = time()   
                add_time('var_cds',te - ts)
            except Exception as e:
                print('name: %s \nref: %s\npep: %s\n%s\n' % (scan_name,ref_prot,peptide,e), file=sys.stderr)
        return pep_cds

    def get_mapping(acc,pep_start,pep_end):
        ts = time()
        p_start = (pep_start - 1) * 3
        p_end = pep_end * 3
        map_params = {"acc" : acc, "p_start" : p_start, "p_end" : p_end}
        if args.debug:
            print('%s' % map_params, file=sys.stderr)
        locs = [l for l in map_cursor.execute(MAP_QUERY,map_params)]
        exons = []
        ##       =========	pep
        ##  ---			continue
        ##      ---		trim
        ##          ---		copy
        ##              ---	trim
        ##                 ---  break
        c_end = 0
        for i, (acc,chrom,start,end,strand,cds_start,cds_end) in enumerate(locs):
            if args.debug:
                print('Prot: %s\t%s:%d-%d\t%s\t%d\t%d' % (acc,chrom,start,end,strand,cds_start,cds_end),file=sys.stderr)
            c_start = c_end
            if cds_end < p_start:
                continue
            if cds_start >= p_end:
                break
            if strand == '+':
                if cds_start < p_start:
                    start += p_start - cds_start
                if cds_end > p_end:
                    end -= cds_end - p_end
            else:
                if cds_start < p_start:
                    end -= p_start - cds_start
                if cds_end > p_end:
                    start += cds_end - p_end
            c_end = c_start + abs(end - start)
            if args.debug:
                print('Pep:  %s\t%s:%d-%d\t%s\t%d\t%d' % (acc,chrom,start,end,strand,cds_start,cds_end),file=sys.stderr)
            exons.append([acc,chrom,start,end,strand,c_start,c_end])
        te = time()   
        add_time('get_mapping',te - ts)
        return exons 

    def get_cds(exons):
        ts = time()
        seqs = []
        for i, (acc,chrom,start,end,strand,cds_start,cds_end) in enumerate(exons):
            seq = get_sequence(chrom, min(start,end), max(start,end))
            if strand == '-': 
                seq = reverse_complement(seq)
            seqs.append(seq)
        te = time()   
        add_time('get_cds',te - ts)
        if args.debug:
            print('CDS:  %s' % str(seqs),file=sys.stderr)
        return ''.join(seqs) if seqs else ''

    def genomic_mapping_count(peptide):
        ts = time()
        params = {"sequence" : peptide}
        acc_locs = [l for l in mz_cursor.execute(PEPTIDE_ACC_QUERY,params)]
        te = time()   
        add_time('PEPTIDE_ACC_QUERY',te - ts)
        if acc_locs:
            if len(acc_locs)  == 1:
                return 1
            locations = set()
            for i,acc_loc in enumerate(acc_locs):
                (acc,pep_start,pep_end) = acc_loc            
                if acc in unmapped_accs:
                    continue
                try:
                    add_time('GENOMIC_POS_QUERY_COUNT',1)
                    ts = time()
                    p_start = pep_start * 3
                    p_end = pep_end * 3
                    params = {"acc" : acc, "cds_offset" : p_start}
                    (start_chrom,start_pos) = map_cursor.execute(GENOMIC_POS_QUERY, params).fetchone()
                    params = {"acc" : acc, "cds_offset" : p_end}
                    (end_chrom,end_pos) = map_cursor.execute(GENOMIC_POS_QUERY, params).fetchone()
                    locations.add('%s:%s-%s:%s' % (start_chrom,start_pos,end_chrom,end_pos))
                    te = time()   
                    add_time('GENOMIC_POS_QUERY',te - ts)
                except:
                    unmapped_accs.add(acc)
                    print('Unmapped: %s' % acc, file=sys.stderr)
            return len(locations)
        return -1
        
    def spectrum_peptide_count(spectrum_id):
        ts = time()
        params = {"sr_id" : spectrum_id}
        pep_count = mz_cursor.execute(SPECTRUM_PEPTIDES_QUERY, params).fetchone()[0]
        te = time()   
        add_time('SPECTRUM_PEPTIDES_QUERY',te - ts)
        return pep_count

    def get_exon_pileup(chrom,chromStart,chromEnd):
        cols = []
        for pileupcolumn in samfile.pileup(chrom, chromStart, chromEnd):
            if chromStart <= pileupcolumn.reference_pos <= chromEnd:
                bases = dict()
                col = {'depth' : 0, 'cov' : pileupcolumn.nsegments, 'pos': pileupcolumn.reference_pos, 'bases' : bases}
                for pileupread in pileupcolumn.pileups:
                    if not pileupread.is_del and not pileupread.is_refskip:
                        col['depth'] += 1
                        base = pileupread.alignment.query_sequence[pileupread.query_position]
                        if base not in bases:
                            bases[base] = 1
                        else:
                            bases[base] += 1
                cols.append(col)
        return cols

    codon_map = {"TTT":"F", "TTC":"F", "TTA":"L", "TTG":"L",
                 "TCT":"S", "TCC":"S", "TCA":"S", "TCG":"S",
                 "TAT":"Y", "TAC":"Y", "TAA":"*", "TAG":"*",
                 "TGT":"C", "TGC":"C", "TGA":"*", "TGG":"W",
                 "CTT":"L", "CTC":"L", "CTA":"L", "CTG":"L",
                 "CCT":"P", "CCC":"P", "CCA":"P", "CCG":"P",
                 "CAT":"H", "CAC":"H", "CAA":"Q", "CAG":"Q",
                 "CGT":"R", "CGC":"R", "CGA":"R", "CGG":"R",
                 "ATT":"I", "ATC":"I", "ATA":"I", "ATG":"M",
                 "ACT":"T", "ACC":"T", "ACA":"T", "ACG":"T",
                 "AAT":"N", "AAC":"N", "AAA":"K", "AAG":"K",
                 "AGT":"S", "AGC":"S", "AGA":"R", "AGG":"R",
                 "GTT":"V", "GTC":"V", "GTA":"V", "GTG":"V",
                 "GCT":"A", "GCC":"A", "GCA":"A", "GCG":"A",
                 "GAT":"D", "GAC":"D", "GAA":"E", "GAG":"E",
                 "GGT":"G", "GGC":"G", "GGA":"G", "GGG":"G",}

    aa_codon_map = dict()
    for c,a in codon_map.items():
        aa_codon_map[a] = [c] if a not in aa_codon_map else aa_codon_map[a] + [c]

    aa_na_map =  dict()   # m[aa]{bo : {b1 : [b3]
    for c,a in codon_map.items():
        if a not in aa_na_map:
            aa_na_map[a] = dict()
        d = aa_na_map[a]
        for i in range(3):
            b = c[i]
            if i < 2:
                if b not in d:
                    d[b] = dict() if i < 1 else set()
                d = d[b]
            else:
                d.add(b)

    def get_pep_codon(pileup, idx, aa, ao):
        try:
            ts = time()
            bases = []
            for i in range(3):
                if i < ao:
                    bases.append(list(set([c[i] for c in aa_codon_map[aa]])))
                else:
                    bases.append([b for b, cnt in reversed(sorted(pileup[idx + i]['bases'].iteritems(), key=lambda (k,v): (v,k)))])
                print('%s' % bases)
            for b0 in bases[0]:
                if b0 not in aa_na_map[aa]:
                    continue
                for b1 in bases[1]:
                    if b1 not in aa_na_map[aa][b0]:
                        continue
                    for b2 in bases[2]:
                        if b2 in aa_na_map[aa][b0][b1]:
                            return '%s%s%s' % (b0,b1,b2)
            te = time()   
            add_time('pep_codon',te - ts)
        except Exception as e:
            print("get_pep_codon: %s %s %s %s"
                      % (aa, ao, idx, pileup), file=sys.stderr)
            raise e
        return None  

    def write_probed(chrom,chromStart,chromEnd,strand,blockCount,blockSizes,blockStarts,
                     spectrum,protacc,peptide,uniqueness,genomeReference,score=1000,
                     psmScore='.', fdr='.', mods='.', charge='.',
                     expMassToCharge='.', calcMassToCharge='.',
                     psmRank='.', datasetID='.', uri='.'):
        probed.write('%s\t%d\t%d\t%s\t%d\t%s\t%d\t%d\t%s\t%d\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\n' % \
            (chrom,chromStart,chromEnd,spectrum,score,strand,chromStart,chromEnd,'0',blockCount,
             ','.join([str(v) for v in blockSizes]),
             ','.join([str(v) for v in blockStarts]),
             protacc,peptide,uniqueness, genomeReference,
             psmScore, fdr, mods, charge, expMassToCharge, calcMassToCharge, psmRank, datasetID, uri))

    def get_genomic_location(exons):
        chrom = exons[0][1]
        strand = exons[0][4]
        pos = [exon[2] for exon in exons] + [exon[3] for exon in exons]
        chromStart = min(pos)
        chromEnd = max(pos)
        blockCount = len(exons)
        blockSizes = [abs(exon[3] - exon[2]) for exon in exons]
        blockStarts = [min(exon[2],exon[3])  - chromStart for exon in exons]
        return (chrom,chromStart,chromEnd,strand,blockCount,blockSizes,blockStarts)

    def get_psm_modifications(peptide_ref):
        mods = []
        ts = time()
        params = {"peptide_ref" : peptide_ref}
        pepmods = [m for m in mz_cursor.execute(PEP_MODS_QUERY, params)]
        if pepmods:
            for (location, residue, name, modType, unimod) in pepmods:
                mods.append('%s-%s' % (location, unimod if unimod else '%s%s' % (name,residue)))
        te = time()
        add_time('PEP_MODS_QUERY',te - ts)
        return ';'.join(mods)


    """
    QNAME
    FLAG
    RNAME
    POS
    CIGAR
    SEQ
    'NH' : 'i', #number of genomic locations to which the peptide sequence maps
    'XO' : 'Z', #uniqueness of the peptide mapping
    'XL' : 'i', #number of peptides to which the spectrum maps
    'XP' : 'Z', #peptide sequence
    'YP' : 'Z', #Protein accession ID from the original search result
    'XF' : 'Z', #Reading frame of the peptide (0, 1, 2)
    'XI' : 'f', #Peptide intensity
    'XB' : 'Z', #massdiff; experimental mass; calculated mass massdiff can be calculated by experimental mass - calculated mass. If any number is unavailable, the value should be left blank (such as 0.01;;).
    'XR' : 'Z', #reference peptide sequence
    'YB' : 'Z', #Preceding amino acids (2 AA, B stands for before).
    'YA' : 'Z', #Following amino acids (2 AA, A stands for after).
    'XS' : 'f', #PSM score
    'XQ' : 'f', #PSM FDR (i.e. q-value or 1-PEP).
    'XC' : 'i', #peptide charge
    'XA' : 'i', #Whether the peptide is annotated 0:yes; 1:parially unknown; 2:totally unknown;
    'XM' : 'Z', #Modifications
    'XN' : 'i', #Number of missed cleavages in the peptide (XP)
    'XT' : 'i', #Enzyme specificity
    'XE' : 'i', #Enzyme used in the experiment
    'XG' : 'A', #Peptide type
    'XU' : 'Z', #URI
    """
    psm_cursor = get_connection(args.mzsqlite_file).cursor()
    ts = time()
    psms = psm_cursor.execute(PSM_QUERY)
    te = time()   
    add_time('PSM_QUERY',te - ts)
    proBAM = ProBAM(species=None,assembly=args.genomeReference,seqlens=seqlens,comments=[])
    proBED = ProBED(species=None,assembly=args.genomeReference,comments=[])
    for i, psm in enumerate(psms):
        probam_dict = PROBAM_DEFAULTS.copy()
        (acc,pep_start,pep_end,aa_pre,aa_post,peptide,spectrum_id,spectrum_title,rank,charge,calcmass,exprmass,pepref) = psm
        scan_name = spectrum_title if spectrum_title else spectrum_id
        if args.debug:
            print('\nPSM: %d\t%s' % (i, '\t'.join([str(v) for v in (acc,pep_start,pep_end,peptide,spectrum_id,scan_name,rank,charge,calcmass,exprmass)])), file=sys.stderr)
        exons = get_mapping(acc,pep_start,pep_end)
        if args.debug:
            print('%s' % exons, file=sys.stderr)
        if not exons:
            continue
        mods = get_psm_modifications(pepref)
        (chrom,chromStart,chromEnd,strand,blockCount,blockSizes,blockStarts) = get_genomic_location(exons)
        ref_cds = get_cds(exons)
        if args.debug:
            print('%s' % ref_cds, file=sys.stderr)
        ref_prot = translate(ref_cds)
        if args.debug:
            print('%s' % ref_prot, file=sys.stderr)
            print('%s' % peptide, file=sys.stderr)
        spectrum_peptides = spectrum_peptide_count(spectrum_id)
        peptide_locations = genomic_mapping_count(peptide)
        if args.debug:
            print('spectrum_peptide_count: %d\tpeptide_location_count: %d' % (spectrum_peptides,peptide_locations), file=sys.stderr)
        uniqueness = 'unique' if peptide_locations == 1 else 'not-unique[unknown]'
        ts = time()
        proBEDEntry = ProBEDEntry(chrom,chromStart,chromEnd,
                                  '%s_%s' % (acc,scan_name),
                                  1000,strand,
                                  blockCount,blockSizes,blockStarts,
                                  acc,peptide,uniqueness,args.genomeReference,
                                  charge=charge,expMassToCharge=exprmass,calcMassToCharge=calcmass,
                                  mods=mods if mods else '.', psmRank=rank)
        proBED.add_entry(proBEDEntry)
        te = time()   
        add_time('add_probed',te - ts)
        if len(ref_prot) != len(peptide):
            continue
        ts = time()
        probam_dict['NH'] = peptide_locations
        probam_dict['XO'] = uniqueness 
        probam_dict['XL'] = peptide_locations 
        probam_dict['XP'] = peptide
        probam_dict['YP'] = acc
        probam_dict['XC'] = charge
        probam_dict['XB'] = '%f;%f;%f' % (exprmass - calcmass, exprmass, calcmass)
        probam_dict['XR'] = ref_prot  # ? dbSequence
        probam_dict['YA'] = aa_post
        probam_dict['YB'] = aa_pre
        probam_dict['XM'] = mods if mods else '*'
        flag = 16 if strand == '-' else 0
        if str(rank)!=str(1) and rank!='*' and rank!=[] and rank!="":
            flag += 256
        probam_dict['XF'] = ','.join([str(e[2] % 3) for e in exons])
        ## check for variation from ref_cds
        pep_cds = get_variant_cds(exons,ref_prot,peptide,ref_cds)
        peptide_type = '*'
        ## XG classify peptide
        probam_dict['XG'] = get_peptide_type(exons)
        ## probam_dict['MD'] = peptide

        ## FIX  SAM sequence is forward strand
        seq = pep_cds if strand == '+' else reverse_complement(pep_cds)
        ## cigar based on plus strand
        cigar = ''
        if strand == '+':
            blkStarts = blockStarts
            blkSizes = blockSizes
        else:
            blkStarts = [x for x in reversed(blockStarts)]
            blkSizes = [x for x in reversed(blockSizes)]
        for j in range(blockCount):
            if j > 0:
                 intron = blkStarts[j] - (blkStarts[j-1] + blkSizes[j-1])
                 if intron > 0:
                     cigar += '%dN' % intron
            cigar += '%dM' % blkSizes[j]
        ## Mods TODO
        proBAMEntry = ProBAMEntry(qname=scan_name, flag=flag, rname=chrom, pos=chromStart+1,
                                  cigar=cigar,seq=seq,optional=probam_dict)
        proBAM.add_entry(proBAMEntry)
        te = time()   
        add_time('add_probam',te - ts)
        
        if args.debug:
            print('%s' % probam_dict, file=sys.stderr)

        if args.limit and i >= args.limit: 
            break
    if args.probed:
        ts = time()
        with open(args.probed,'w') as fh:
            proBED.write(fh)
        te = time()   
        add_time('write_probed',te - ts)
    if args.prosam or args.probam:
        samfile = args.prosam if args.prosam else 'temp.sam'
        ts = time()
        with open(samfile,'w') as fh:
            proBAM.write(fh)
        te = time()   
        add_time('write_prosam',te - ts)
        if args.probam:
            ts = time()
            bamfile = args.prosam.replace('.sam','.bam')
            pysam.view(samfile, '-b', '-o', args.probam, catch_stdout=False)
            te = time()   
            add_time('write_probam',te - ts)
            pysam.index(args.probam)

    print('\n%s\n' % str(timings), file=sys.stderr)

if __name__ == "__main__":
    __main__()