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-   -   compare genomic features from gff file to a SAM/BED file (http://seqanswers.com/forums/showthread.php?t=31414)

jazz 06-26-2013 07:26 AM

compare genomic features from gff file to a SAM/BED file
 
I have an alignment from Bowtie2 for my RNA-IP and I want to compile a pie chart of % of reads mapping to different genomic features like exons, introns, UTR's etc. I am planning to compare my BAM/BED file to a genome annotation gff file. I have heard of intersectbed in bedtools, however the gff files and bed files have genomic coordinates in different columns. can anyone suggest a simple fix for that? I have also heard of people using mpileup option in samtools, although I have no idea how that would work here. I am a bioinformatics newbie, so writing custom scripts is out of question at this point. Given that this is a very commonly sought after problem, I am hopeful that one of the great minds would have found an easy solution for this.
Thanks in advance!

dariober 06-26-2013 03:06 PM

Quote:

Originally Posted by jazz (Post 108784)
I have heard of intersectbed in bedtools, however the gff files and bed files have genomic coordinates in different columns. can anyone suggest a simple fix for that?

No need to fix anything! Bedtools recognizes the file format (GFF/bed/bam) and handles it accordingly. Have you tried it at all with your files?

Dario

jazz 07-01-2013 08:32 AM

I am getting a bizarre result from using the intersectbed from bedtools. The output bed file contains strange characters and not the chromosomal coordinates as I expected.
Any suggestions?
Can anyone explain if the samtools mpileup would help?

Thanks

dariober 07-01-2013 08:53 AM

Quote:

Originally Posted by jazz (Post 109197)
I am getting a bizarre result from using the intersectbed from bedtools. The output bed file contains strange characters and not the chromosomal coordinates as I expected.
Any suggestions?
Can anyone explain if the samtools mpileup would help?

Thanks

Hi there,

It would help other people answering you if you post
- The command line you executed
- A sample (a few lines) of the output including errors and messages
- A sample of the input files

Dario

jazz 07-01-2013 08:59 AM

Sorry!

Here is the command I tried:

intersectbed -a Inputfile.bed -b compare.gff > output.bed

The first few lines of the resulting bed file look like this:

BAM◊‹%@HD VN:1.0 SO:unsorted
@SQ SN:scaffold16 LN:1343
@SQ SN:scaffold6 LN:1021
@SQ SN:scaffold4 LN:958
@SQ SN:scaffold22 LN:923

Below, there is something like this:

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#%!%&'%%#%%ASCXNCXMCXOCXGCNMCMDZ25YTZUU”)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2201:12566:71306ABAA%&$)())('% "%%#%'%%%%%"!ASCXNCXMCXOCXGCNMCMDZ25YTZUU”)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2202:18198:47631ABAA(()(&()()'&'&''%$%###!!ASCXNCXMCXOCXGCNMCMDZ24YTZUU”(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2202:2362:66940ABAA())))))))))'''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ24YTZUU”(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2203:2172:23622@ABAA%'&()))'&&''%%%%%""ASCXNCXMCXOCXGCNMCMDZ20YTZUU”(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2205:4395:36383pABAA()))))))()'''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ23YTZUU”)IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2216:10710:74034pABAA&&&$'(&'%" #'#'$#%%#ASc˚XNCXMCXOCXGCNMCMDZ0A22YTZUU‘(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1105:3277:44016($!$(D%"'$$''$"$' ''%%$%##ASCXNCXMCXOCXGCNMCMDZ24YTZUU‘(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1208:7802:34643`($!$(D(&$$)($&&''&''%%%%%ASCXNCXMCXOCXGCNMCMDZ22YTZUU‘(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2116:7778:29884`($!$(D))))))))('''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ22YTZUU‘(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2201:2018:63939P($!$(D)'&&'((#'%#%'#%%%%"""ASCXNCXMCXOCXGCNMCMDZ21YTZUU‘(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2205:6440:23480@($!$(D)))))))'''''%%%%#"!!ASCXNCXMCXOCXGCNMCMDZ20YTZUU’)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1104:10597:245130ABAA))))))'''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ19YTZUU’)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1107:11301:46632@ABAA))))))('%''&%%%%%""!ASCXNCXMCXOCXGCNMCMDZ20YTZUU’(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1116:7714:37811PABAA!&(!((&" ''%'%#%%%"""ASCXNCXMCXOCXGCNMCMDZ21YTZUU’)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1201:14408:333510ABAA))))))'''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ19YTZUU’)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1212:15336:85329PABAA%$(!$&&!%##%%%%##ASCXNCXMCXOCXGCNMCMDZ21YTZUU’(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1213:8794:577530ABAA())(()%''!##%%%#ASCXNCXMCXOCXGCNMCMDZ19YTZUU◊(*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2208:8325:13729PABAA)))))(()'''''%%%%%"""ASCXNCXMCXOCXGCNMCMDZ21YTZUU€) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2103:3275:100501PBAA"%&)))(&''&''%%%#%"ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUU›) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2104:18900:88114PAA ""%%%%%'''''))())))(ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUU›) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1112:15391:75683AA(B))))))))))))'''''%%%%%"""ASCXSc˙XNCXMCXOCXGCNMCMDZ25YTZUU›)"IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1211:21140:394260AA')()))'''''!%%%%""ASCXSc˚XNCXMCXOCXGCNMCMDZ19YTZUUfi( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1209:7509:61608P$(DAB""%%%%%'&&'')))))())ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUUfi)#IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1101:19285:54092@$(DAB(&())((''''%%%%%%"ASCXSc˙XNCXMCXOCXGCNMCMDZ20YTZUU) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1106:17172:28460PA(B%#$#%''''&)))()))&ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUU)"IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1214:13003:11554@A(B"""%%%%%'''''))()))(ASCXSc˚XNCXMCXOCXGCNMCMDZ20YTZUU( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2213:6335:24825PA(B"""%%%%%'''&'))))))))ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUU (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1212:7719:35597@((B #$%%' %'&'(&!!&&(ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2105:12730:197780((B""%%%%%%''''&((&&'ASCXSCXNCXMCXOCXGCNMCMDZ19YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1104:10693:56496P!(AD( """%%%%%''''&))()))))ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1209:14997:555950!(AD("""%%%%%'''''(())))ASCXSCXNCXMCXOCXGCNMCMDZ19YTZUU (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2110:11871:3278P!(AD( """%%%%#'''''()')))))ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2114:18299:89262P!(AD( #####'''!%$&(&&ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2206:13913:17841P!(AD( """%%%%#'''''))))))))ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2206:20333:22711@!(AD("""%%%%#'%'''()(()))ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUU (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2106:4675:75715@"!!)))()))'''''%%#%%"!"ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUU< )"IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2101:16869:10135 BD(H"B)(((&'!'!&%%#%%ASCXSc˚XNCXMCXOCXGCNMCMDZ18YTZUU@ ) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2213:17512:13789PD(H"B!B ""%%#%%'''''()))))))ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUUG )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1104:16569:86225@$"$!B#"'#'&"#%##!#ASc˚XSc˚XNCXMCXOCXGCNMCMDZ11A8YTZUUG (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1116:5607:77564@$"!!B&&($(((%''###%###ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUUG )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1203:10461:40370`$"!!B&%#$!&&%%''!##%%%%ASCXSCXNCXMCXOCXGCNMCMDZ22YTZUUG (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2102:8390:14238@$"!!B(&&%(#'#%##'#%%%%ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUUO )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1206:18423:84555`!!B!B('$'%('"$&&##' %###ASCXSCXNCXMCXOCXGCNMCMDZ22YTZUUT (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1103:3897:51516@!B("("H)))))))'''''%%%%%"""ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUUT )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2105:19766:933400!B("("@((%))(%%%'&###%%"ASCXSCXNCXMCXOCXGCNMCMDZ19YTZUUi ) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2115:20101:40407pH(B(DB""%%%%%'&%&'%((&'&((((ASCXSc˚XNCXMCXOCXGCNMCMDZ23YTZUUr *IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:10371:100573P!BH(H(@&&&$!$(&'&'''%%%%%"""ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUU ("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2211:1274:32249@!BB!((&%()&%''&'%%%##!!ASCXSc˚XNCXMCXOCXGCNMCMDZ20YTZUU≠ ) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1103:20055:27875!H"!((''())))(('''''%%%%%!!ASCXSc˚XNCXMCXOCXGCNMCMDZ24YTZUU )"IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2206:11020:48210 "!!)))))'''''%%%%%!!!ASCXSc˚XNCXMCXOCXGCNMCMDZ18YTZUU (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2115:12981:3475`"(((B&&&'&'$ %%!'%#%##ASCXSCXNCXMCXOCXGCNMCMDZ22YTZUU )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2116:11206:58814@"A&$(&&&)'''%'%%%%%"""ASc˚XSc˙XNCXMCXOCXGCNMCMDZ0T19YTZUU
)*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1206:19932:98308p$H!!"""%%%%%''''')())))))))ASCXNCXMCXOCXGCNMCMDZ23YTZUU
("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1109:1943:983830!!""""%%%%%'''''))))()ASCXSc˚XNCXMCXOCXGCNMCMDZ19YTZUU
("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2108:7758:743890!!""""%%%%%'''''))))))ASCXSc˚XNCXMCXOCXGCNMCMDZ19YTZUU
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:2705:390750B!!"## ""#%$ &ASc˚XNCXMCXOCXGCNMCMDZ0T18YTZUU"
)IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2213:20007:56613"!$###!%##%&'$%$&&ASCXSCXNCXMCXOCXGCNMCMDZ24YTZUU.
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1204:5967:66521@!$A))(('))'''''%%%%#!!ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUU?
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:6446:99235@""AH(())))(''#&'%%%%%"""ASc˙XSc˙XNCXMCXOCXGCNMCMDZ0C19YTZUU^
)IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2106:11632:21600@($")((((()'&'''%%%%%"""ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUU_
( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2212:8365:29810†HH(!"######%''%((&'&$'&(&'$&ASCXSc˚XNCXMCXOCXGCNMCMDZ26YTZUUs
( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1113:5678:26750P"B(%#%%%'&%%'($ $!#'ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUUz

These are just snippets, since the whole file is pretty big

jazz 07-01-2013 09:04 AM

I tried it again and this time it gave a sane output like this

scaffold68 3144 3165 JLK5VL1:383:H0TDTADXX:1:1116:17468:54631 42 +
scaffold68 3162 3184 JLK5VL1:383:H0TDTADXX:1:1205:10063:18561 42 -
scaffold68 3213 3232 JLK5VL1:383:H0TDTADXX:1:2211:8864:24912 42 -
scaffold68 3229 3250 JLK5VL1:383:H0TDTADXX:1:2103:11115:20978 42 -
scaffold68 3230 3250 JLK5VL1:383:H0TDTADXX:1:2210:10068:78079 23 -
scaffold68 3325 3346 JLK5VL1:383:H0TDTADXX:1:2204:21149:40214 24 +
scaffold68 3352 3373 JLK5VL1:383:H0TDTADXX:1:2114:18637:46559 24 -
scaffold68 3352 3373 JLK5VL1:383:H0TDTADXX:1:2216:4275:35276 42 -
scaffold68 3367 3389 JLK5VL1:383:H0TDTADXX:1:1102:12483:97822 24 +
scaffold68 3405 3427 JLK5VL1:383:H0TDTADXX:1:1215:17603:90243 42 -

lran2008 11-04-2013 08:38 AM

Hi, have you found out how to do it? I am also having this problem.

shi 11-04-2013 01:31 PM

You may try the featureCounts program included in the Subread package.

dariober 11-05-2013 12:04 AM

Quote:

Originally Posted by jazz (Post 109210)
Here is the command I tried:

intersectbed -a Inputfile.bed -b compare.gff > output.bed

The first few lines of the resulting bed file look like this:

BAM◊%@HD VN:1.0 SO:unsorted
@SQ SN:scaffold16 LN:1343
@SQ SN:scaffold6 LN:1021
@SQ SN:scaffold4 LN:958
@SQ SN:scaffold22 LN:923

Hi- I suspect your "Inputfile.bed" is not a bed file as the name suggests but a bam file. Could you post the output of:

Code:

head Inputfile.bed
head compare.gff

If Inputfile.bed is indeed a bam file you should use something like (depending on exactly what output you want):
Code:

intersectBed -abam Inputfile.bam -b compare.gff -bed -wb > results.bed


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