compare genomic features from gff file to a SAM/BED file

[jazz]

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]

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]

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]

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]

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

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*IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:10371:100573P!BÑHÑ(HÑ(@&&&$!$(&'&'''%%%%%"""ASCXSCXNCXMCXOCXGCNMCMDZ21YTZUUíü ("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2211:1274:32249@!BÑBÑàÑ!((&%()&%''&'%%%##!!ASCXSc˚XNCXMCXOCXGCNMCMDZ20YTZUUô≠ ) IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1103:20055:27875ÄÑ!ÇÑH"Ç!Ç((''())))(('''''%%%%%!!ASCXSc˚XNCXMCXOCXGCNMCMDZ24YTZUUêª )"IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2206:11020:48210 "Ç!Çà!Ç)))))'''''%%%%%!!!ASCXSc˚XNCXMCXOCXGCNMCMDZ18YTZUUï¿ (IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2115:12981:3475`"(Å(Ç(Bà&&&'&'$ %%!'%#%##ASCXSCXNCXMCXOCXGCNMCMDZ22YTZUUï )IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2116:11206:58814@"ÅAàÅ&$(&&&)'''%'%%%%%"""ASc˚XSc˙XNCXMCXOCXGCNMCMDZ0T19YTZUUî
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("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1109:1943:983830ÇÑ!!Å"àÑ"""%%%%%'''''))))()ASCXSc˚XNCXMCXOCXGCNMCMDZ19YTZUUë
("IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2108:7758:743890ÇÑ!!Å"àÑ"""%%%%%'''''))))))ASCXSc˚XNCXMCXOCXGCNMCMDZ19YTZUUè
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:2705:390750BÑ!!Å"àÑ## ""#%$ &ASc˚XNCXMCXOCXGCNMCMDZ0T18YTZUUô"
)IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2213:20007:56613ÄÅ"àÑ!ÑÅ$###!%##%&'$%$&&ASCXSCXNCXMCXOCXGCNMCMDZ24YTZUUí.
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1204:5967:66521@!ÑÅ$AÇ))(('))'''''%%%%#!!ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUUî?
(IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2111:6446:99235@"à"àAàÑH(())))(''#&'%%%%%"""ASc˙XSc˙XNCXMCXOCXGCNMCMDZ0C19YTZUUì^
)IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2106:11632:21600@(Ñà$Çà")((((()'&'''%%%%%"""ASCXSCXNCXMCXOCXGCNMCMDZ20YTZUUõ_
( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:2212:8365:29810†àHÅÇH(Ç!ÅàÅ"######%''%((&'&$'&(&'$&ASCXSc˚XNCXMCXOCXGCNMCMDZ26YTZUUîs
( IˇˇˇˇˇˇˇˇJLK5VL1:383:H0TDTADXX:1:1113:5678:26750PàÅ"BÑ(Ä%#%%%'&%%'($ $!#'ASCXSc˚XNCXMCXOCXGCNMCMDZ21YTZUUîz

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

[jazz]

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]

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

[shi]

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

[dariober]

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:

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):

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