Hi,Paul Korir:
   Thank you for yours help.I have known the reason,But I also I have a little 
problem about to solve the question.
   if I want to add a XS tag ,what should I do ,can you tell me in detail(like 
that ,dose it only have two value ,such as XS:A:-,XS:A:+ ,not have 
XS:B([B-Z]):+ ? 
Best wishes
----- 原始邮件 -----
发件人: "Paul Korir" <polaris...@gmail.com>
收件人: "lishiyong" <lishiy...@genomics.org.cn>
抄送: "tophat.cufflinks" <tophat.cuffli...@gmail.com>, "galaxy-user" 
<galaxy-user@lists.bx.psu.edu>, "高欢" <gaoh...@genomics.org.cn>
发送时间: 星期一, 2011年 4 月 11日 下午 11:10:56
主题: Re: [galaxy-user] cufflinks FPKM problem

Hi Li, 

Tophat includes a custom tag 'XS' at the end of spliced read alignments which 
your pipeline is not aware about. 

The following is taken from http://cufflinks.cbcb.umd.edu/manual.html 


"Cufflinks takes a text file of SAM alignments as input. For more details on 
the SAM format, see the specification . The RNA-Seq read mapper TopHat produces 
output in this format, and is recommended for use with Cufflinks. However 
Cufflinks will accept SAM alignments generated by any read mapper. Here's an 
example of an alignment Cufflinks will accept: 
s6.25mer.txt-913508     16      chr1 4482736 255 14M431N11M * 0 0 \ 
CAAGATGCTAGGCAAGTCTTGGAAG IIIIIIIIIIIIIIIIIIIIIIIII NM:i:0 XS:A:- 
Note the use of the custom tag XS . This attribute, which must have a value of 
"+" or "-", indicates which strand the RNA that produced this read came from. 
While this tag can be applied to any alignment, including unspliced ones, it 
must be present for all spliced alignment records (those with a 'N' operation 
in the CIGAR string)." 

Kind regards, 

Paul 



2011/4/11 lishiyong < lishiy...@genomics.org.cn > 




Hi: 
I use the solid PE sequencing data and mapped with the bioscope tools(AB 
company supported) ,which is better for solid data mapping ,so I don't use the 
bowtie to map . Igain the BAM file! Now ,I want use the cufflinks to calculate 
the gene expression. But there is a error. 

[15:08:06] Inspecting reads and determining fragment length distribution. 
BAM record error: found spliced alignment without XS attribute 
BAM record error: found spliced alignment without XS attribute 
 the BAM file : 
323_358_2010    73      chr1    343     0       45M5H   *       0       0       CCCTAACCCTACCCTAACCCTAACCCTAACCCTAACCCTAACCCT   IIIIIIIIIII))C/1<DE''@DAHD379AID1;7BI+'7))I?3   RG:Z:20110328192522421   NH:i:0  CM:i:4  SM:i:2  CQ:Z:A=ABA<<>@?<4)='))415'-4118-'1)9>'+1'<6+'1)85+)-+6- CS:Z:T20023010023110230100030100230100230100030000200000
 
423_236_1955    81      chr1    550     0       8H42M   =       699451  698945  GTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCTCTCCGG      GF>IIII%%III))8IIII?IIII%%IIIIIIIIIIIIIIII      RG:Z:20110328192522421   NH:i:2  CM:i:5  SM:i:3  CQ:Z:9BA<AAB>;?AB:55;A%9?AB,4:@@*/)7>2<%5@<:3,;-.%8.*;5 CS:Z:T20302222311033322303302232133302223222131122330223
 
298_1884_1495   113     chr1    562     0       7H43M   chr3    199392032       0       ACGCAGCTCCGCCCTCGCGGTGCTCTCCGGGTCTGTGCTGAGG     5AI;6:>AIIII>?I7FIEIIIIIIIIIIIIIIIIIIIIIIII      RG:Z:20110328192522421  NH:i:2  CM:i:0  SM:i:3  CQ:Z:BB@7<AB8@ABA=2;=>82:?A388.A&28(77;64.1*-/<&0:9/%3? CS:Z:T20221231112210030222231103332200330223213312222022
 
62_1428_1954    89      chr1    562     1       50M     *       0       0       ACGCAGCTCCGCCCTCGCGGTGCTCTCCGGGTCTGTGCTGAGGAGAATGC      *=AIII4/CII=%%I((=EIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII       RG:Z:20110328192522421  NH:i:0  CM:i:4  SM:i:0  CQ:Z:@B@BABB=ABBB?@A=B>>@@?<;?>B>=<??'7(;A%&849+%0:@.4* CS:Z:T13130222022123111221003022223110331222033022321331
 

  
I have sorted the bam file and the gtf file. 
cufflinks  -G refGene_hg18.gtf -p 3 -r  human_hg18.fa -o test  test.pe.bam  
(the version of cufflinks is v0.9.2 )  
    Who know the reason ,and what shoud I do! 
best wishes! 
Shiyong Li   
2011-04-11 

lishiyong 
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-- 
Paul Korir 
www.paulkorir.com 

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