I have been attempting to map paired end data from 2x76nt sequencing runs of Solexa. I trim reads using scripts written by myself and others to remove any reads with poor quality and trim away adaptor sequences, then discard reads that are <18 nt after trimming. After running alignments with tophat and uploading to the UCSC genome browser I find that mated pairs align very close to each other but are not paired. When I click on one of the mates the description of the read says "
Read name: HWUSI-EAS1712:4:1:827:1321#0
Position: chr2:25159536-25159568
Band: 2qA3
Genomic Size: 33
Alignment Quality: 255
CIGAR string: 33M (33 (mis)Match)
Tags: NM:0 NH:1 XS:-
Flags: 0x49:
(0x40) Read 1 of pair | (0x08) Mate is unmapped | (0x01) Not properly paired
"
but aligned above was also another read with the same ID
"
Read name: HWUSI-EAS1712:4:1:827:1321#0
Position: chr2:25159535-25159563
Band: 2qA3
Genomic Size: 29
Alignment Quality: 255
CIGAR string: 29M (29 (mis)Match)
Tags: NM:0 NH:1 XS:-
Flags: 0x99:
(0x80) Read 2 of pair | (0x10) Read is on '-' strand | (0x08) Mate is unmapped | (0x01) Not properly paired
"
Why are my mates not paired? Is this due to the fact that the reads in my -1 and -2 fastq files are not in the same order? If that is the problem, can anyone suggest a way to put my reads in the same order, since now after trimming there are differing numbers of reads in the -1 and -2 files??
Thanks for any help you'd be able to offer.
-Mike
Read name: HWUSI-EAS1712:4:1:827:1321#0
Position: chr2:25159536-25159568
Band: 2qA3
Genomic Size: 33
Alignment Quality: 255
CIGAR string: 33M (33 (mis)Match)
Tags: NM:0 NH:1 XS:-
Flags: 0x49:
(0x40) Read 1 of pair | (0x08) Mate is unmapped | (0x01) Not properly paired
"
but aligned above was also another read with the same ID
"
Read name: HWUSI-EAS1712:4:1:827:1321#0
Position: chr2:25159535-25159563
Band: 2qA3
Genomic Size: 29
Alignment Quality: 255
CIGAR string: 29M (29 (mis)Match)
Tags: NM:0 NH:1 XS:-
Flags: 0x99:
(0x80) Read 2 of pair | (0x10) Read is on '-' strand | (0x08) Mate is unmapped | (0x01) Not properly paired
"
Why are my mates not paired? Is this due to the fact that the reads in my -1 and -2 fastq files are not in the same order? If that is the problem, can anyone suggest a way to put my reads in the same order, since now after trimming there are differing numbers of reads in the -1 and -2 files??
Thanks for any help you'd be able to offer.
-Mike
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