Hi folks,
I ran the cufflinks with a plus strand reads, but get a minus gene annotation file. Here is my test sam file
@HD VN:1.0 SO:coordinate
@SQ SN:1 LN:301354135
@SQ SN:2 LN:237068873
@SQ SN:3 LN:232140174
@SQ SN:4 LN:241473504
@SQ SN:5 LN:217872852
@SQ SN:6 LN:169174353
@SQ SN:7 LN:176764762
@SQ SN:8 LN:175793759
@SQ SN:9 LN:156750706
@SQ SN:10 LN:150189435
@PG ID:TopHat VN:2.0.11 CL:/usr/local/bin/tophat -p 15 -G gene.gtf index P1-WS_L1_1.fq P1-WS_L1_2.fq
FCC2B6YACXX:6:2112:15906:8029#GTCCAGAA 161 1 161922 50 90M 6 97320042 0 GTAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACT a__ccceeefggaghfcghd^_Wcffhgf`ddgee``baccccc_bcccdbcccccaaZacZaccaccaZ_aacHW`_ccaaTX[T[[aB AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:28C56A4 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1314:16597:88330#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG b__eeeeegfgfg[fhdhighdgiiiiieghgfedcccacccccccccdb_aaTacacccccaccccacc_]aaaacccc]_caaa_acc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2212:16595:7596#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG bbbeeeeegggggiiiiiiiihihiiiifhiggeacccccccccccccdccccccc_ccacccbcccccccccX^`_abaccccccLT[` AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2216:19234:56575#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG b__eeeeefggggiihihifhiihiiii`ghggecccccccccccccbdcacccccaccccccccccccccccX_acccacccccccccc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1102:7629:5131#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ^__eeecegegggiiiihdhghhihhdgff_ad\aabcccaaXbcbbccW[XaccTY[VaacacccVZaccXHW^aabccc_[aaBBBBB AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1106:2433:36794#GTCCAGAA 161 1 161924 50 90M 6 97320059 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC a^_cZc_cccgced_bcf_dg_eaghh`ffgaaS_abcccca[bccbccaX[WaaaaNTVTZaaaaTV__aa]a^TW`ETO[]aaOLO[b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1213:4750:67646#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC _bbeceeeggggcffffgeghghhif[eafggececccccca`bccccbaX_aacWaccaaaaaccZ__X_aX^`BBBBBBBBBBBBBBB AS:i:-11 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1305:10356:81752#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC _bbeeeeegfegghfhhighiiiihihgfhhgf^ccccccccabcccdcccaccaX_accccacacacc]acT_aaa^ac_acccRXOW^ AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1307:2240:48568#GTCCAGAA 161 1 161924 50 90M 6 97320059 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiiiifhihiiifhihggaecccccccacccccbcaccccaccacccccccccccccX_aaabccccacaTXT`b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1309:18142:34116#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ^^_eeceWcgcgfiiaffadgggdghUT`dgegSTZX^WZ^``b`b`bcTVW[ccTYa[aacZ[]_]a_a]aXWW[W`[]_a[acOOO[^ AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1315:17770:73451#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ___eeecccecea^aedgfhhh_fdhaZ_b_YSX_\a]_ZZ[X_bbcbbTOV[aTOOYRV[acaaacTTZaaOWQXQ`[TTLOT]OXa`b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2101:10239:82397#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiifhhhiiiihghiiggeeccccccccccccdcccccccaccccccccccacccacH[`aacccaccacLX[ab AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2103:14004:23483#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiihihiiiiiighiiggaeccccccccccccdbccccccaccccccccccccccccT[aacccccccccL[[`c AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2114:3405:92937#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggifhiihhhhiiihighiiggceccccccccccccccccccccccccccccccccccccc_cccccccaccccXaccc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1103:5811:64120#ATACTCCA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeeggggghhhhiiihihiihfghihggaabcccccccccccdcccccccaaccccccaccaaccccT^a[ac]a]aaccX[]^b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
===============
the cufflinks result:
1 Cufflinks transcript 161923 162013 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "39129707493.1709060669"; frac "1.000000"; conf_lo "0.000000"; conf_hi "84312801802.157990"; cov "42260.084093";
1 Cufflinks exon 161923 162013 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "39129707493.1709060669"; frac "1.000000"; conf_lo "0.000000"; conf_hi "84312801802.157990"; cov "42260.084093";
I am total confused. Hope you could help.
Thank you in advance.
Best,
Jie
I ran the cufflinks with a plus strand reads, but get a minus gene annotation file. Here is my test sam file
@HD VN:1.0 SO:coordinate
@SQ SN:1 LN:301354135
@SQ SN:2 LN:237068873
@SQ SN:3 LN:232140174
@SQ SN:4 LN:241473504
@SQ SN:5 LN:217872852
@SQ SN:6 LN:169174353
@SQ SN:7 LN:176764762
@SQ SN:8 LN:175793759
@SQ SN:9 LN:156750706
@SQ SN:10 LN:150189435
@PG ID:TopHat VN:2.0.11 CL:/usr/local/bin/tophat -p 15 -G gene.gtf index P1-WS_L1_1.fq P1-WS_L1_2.fq
FCC2B6YACXX:6:2112:15906:8029#GTCCAGAA 161 1 161922 50 90M 6 97320042 0 GTAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACT a__ccceeefggaghfcghd^_Wcffhgf`ddgee``baccccc_bcccdbcccccaaZacZaccaccaZ_aacHW`_ccaaTX[T[[aB AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:28C56A4 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1314:16597:88330#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG b__eeeeegfgfg[fhdhighdgiiiiieghgfedcccacccccccccdb_aaTacacccccaccccacc_]aaaacccc]_caaa_acc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2212:16595:7596#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG bbbeeeeegggggiiiiiiiihihiiiifhiggeacccccccccccccdccccccc_ccacccbcccccccccX^`_abaccccccLT[` AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2216:19234:56575#GTCCAGAA 137 1 161923 50 90M * 0 0 TAGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTG b__eeeeefggggiihihifhiihiiii`ghggecccccccccccccbdcacccccaccccccccccccccccX_acccacccccccccc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:27C56A5 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1102:7629:5131#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ^__eeecegegggiiiihdhghhihhdgff_ad\aabcccaaXbcbbccW[XaccTY[VaacacccVZaccXHW^aabccc_[aaBBBBB AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1106:2433:36794#GTCCAGAA 161 1 161924 50 90M 6 97320059 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC a^_cZc_cccgced_bcf_dg_eaghh`ffgaaS_abcccca[bccbccaX[WaaaaNTVTZaaaaTV__aa]a^TW`ETO[]aaOLO[b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1213:4750:67646#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC _bbeceeeggggcffffgeghghhif[eafggececccccca`bccccbaX_aacWaccaaaaaccZ__X_aX^`BBBBBBBBBBBBBBB AS:i:-11 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1305:10356:81752#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC _bbeeeeegfegghfhhighiiiihihgfhhgf^ccccccccabcccdcccaccaX_accccacacacc]acT_aaa^ac_acccRXOW^ AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1307:2240:48568#GTCCAGAA 161 1 161924 50 90M 6 97320059 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiiiifhihiiifhihggaecccccccacccccbcaccccaccacccccccccccccX_aaabccccacaTXT`b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1309:18142:34116#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ^^_eeceWcgcgfiiaffadgggdghUT`dgegSTZX^WZ^``b`b`bcTVW[ccTYa[aacZ[]_]a_a]aXWW[W`[]_a[acOOO[^ AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1315:17770:73451#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC ___eeecccecea^aedgfhhh_fdhaZ_b_YSX_\a]_ZZ[X_bbcbbTOV[aTOOYRV[acaaacTTZaaOWQXQ`[TTLOT]OXa`b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2101:10239:82397#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiifhhhiiiihghiiggeeccccccccccccdcccccccaccccccccccacccacH[`aacccaccacLX[ab AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2103:14004:23483#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggiiiiihihiiiiiighiiggaeccccccccccccdbccccccaccccccccccccccccT[aacccccccccL[[`c AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:2114:3405:92937#GTCCAGAA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeegggggifhiihhhhiiihighiiggceccccccccccccccccccccccccccccccccccccc_cccccccaccccXaccc AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
FCC2B6YACXX:6:1103:5811:64120#ATACTCCA 137 1 161924 50 90M * 0 0 AGCGACCTCAGCAGCCACGGCGCCGGGTCCGGGTCCGGCCAGATTTACCGCCCCGTGGCGCGCTCCGGCGGGTTCGAGCCGGCGGACTGC bbbeeeeeggggghhhhiiihihiihfghihggaabcccccccccccdcccccccaaccccccaccaaccccT^a[ac]a]aaccX[]^b AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:26C56A6 YT:Z:UU XS:A:- NH:i:1
===============
the cufflinks result:
1 Cufflinks transcript 161923 162013 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "39129707493.1709060669"; frac "1.000000"; conf_lo "0.000000"; conf_hi "84312801802.157990"; cov "42260.084093";
1 Cufflinks exon 161923 162013 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "39129707493.1709060669"; frac "1.000000"; conf_lo "0.000000"; conf_hi "84312801802.157990"; cov "42260.084093";
I am total confused. Hope you could help.
Thank you in advance.
Best,
Jie