[BioC] Rsubread/Rsamtools mappings outside of chromosome ranges
Martin Morgan
mtmorgan at fhcrc.org
Tue Feb 14 23:40:40 CET 2012
On 02/14/2012 02:36 PM, Wei Shi wrote:
> Hi Thomas,
>
> After re-checking the changes we have made before for addressing the
> issue of reads mapped out of the chromosome ranges, I found we
> actually made the decision to allow reads to be mapped out of the
> chromosome ranges, due to the fact that the reference sequences might
> not be complete and reads could originate from outside of the
> reference sequences used for mapping.
>
> So you may take Martin's suggestion to add extra parameters when you
Actually, Herve has patched GenomicRanges (1.7.25) in the development
branch so that ranges outside sequence bounds are now accepted (with a
warning).
Martin
> call 'readGappedAlignments' function to make it work for you. Or
> alternatively you may try the 'featureCounts' function in Rsubread
> package to count the number of reads mapped to each gene, if the
> purpose of your analysis is perform RNA-seq analysis. This function
> works on SAM file, so you do not need to convert it to a BAM file.
> But you will have to provide an Arabidopsis anntation file it as it
> has built-in annotation for mouse and human only at present.
>
> I think it might be better if the 'readGappedAlignments' function
> could allow reads being mapped outside of chromosomal ranges. We have
> been successfully running through a SNP discovery pipeline using
> subread/subjunc, samtools and vcftools on unix command lines, which
> does not complain about the reads mapped outside of the chromosomal
> ranges. I guess many other downstream analysis tools do not require
> mapping locations of reads to be within the chromosomal ranges as
> well.
>
> Hope this helps.
>
> Cheers, Wei
>
>
>
> On Feb 14, 2012, at 11:58 AM, Thomas Girke wrote:
>
>> That's great, thanks a lot!
>>
>> BTW: are there any published/shareable performance test results
>> available comparing subread against other RNA-read to genome
>> aligners such as Tophat with respect to the accuracy of aligning
>> reads across exon/intron junctions?
>>
>> Thanks,
>>
>> Thomas
>>
>> On Mon, Feb 13, 2012 at 11:41:42PM +0000, Wei Shi wrote:
>>> Hi Thomas,
>>>
>>> I have just committed changes to both the release version and the
>>> devel version of Rsubread package to fix the bug of mapping reads
>>> out of chromosomal boundaries. They should be available in 24-36
>>> hours. Please rebuild your index when you rerun your alignment.
>>>
>>> Let me know if the problem persists or you encounter further
>>> problems.
>>>
>>> Cheers, Wei
>>>
>>> On Feb 13, 2012, at 11:12 AM, Thomas Girke wrote:
>>>
>>>> Thanks Martin for your suggestion!
>>>>
>>>> Thomas
>>>>
>>>> On Sun, Feb 12, 2012 at 05:28:21PM +0000, Martin Morgan wrote:
>>>>> On 02/10/2012 07:44 PM, Thomas Girke wrote:
>>>>>> With some Illumina libraries I have been running into
>>>>>> problems importing the read mappings from Rsubread into
>>>>>> Rsamtools. After some testing I found out that some reads
>>>>>> reported in Rsubread's SAM output have their end positions
>>>>>> outside of the chromosome ranges. If those reads are
>>>>>> removed from the SAM file then the import into Rsamtools
>>>>>> works just fine. Below is a reproducible example of this
>>>>>> problem.
>>>>>>
>>>>>> I could think of several solutions to fix this, e.g. not
>>>>>> reporting reads outside of chromosome ranges or updating
>>>>>> their length in the CIAGR string. An additional option
>>>>>> could be to remove invalid mappings during the import into
>>>>>> Rsamtools.
>>>>>
>>>>> Hi Thomas --
>>>>>
>>>>> for the latter and for the case where those reads are
>>>>> intrinsically not interesting, it might be reasonable to
>>>>> specify a range on readGappedAlignments that precludes the
>>>>> possibility of extension beyond sequence ends.
>>>>>
>>>>>> si<- seqinfo(BamFile(fl)) gr<- GRanges(seqnames(si),
>>>>>> IRanges(34, seqlengths(si)-34)) bam<-
>>>>>> readGappedAlignments(fl, param=ScanBamParam(which=gr))
>>>>>
>>>>> One would like a better solution, either doing this
>>>>> automatically with a warning, or warning rather than stopping
>>>>> on reads overlapping ends.
>>>>>
>>>>> Martin
>>>>>
>>>>>>
>>>>>> Thanks,
>>>>>>
>>>>>> Thomas
>>>>>>
>>>>>> ################################ ## Read mapping with
>>>>>> Rsubread ## ################################
>>>>>> library(Rsubread) ## Reference source:
>>>>>> ftp://ftp.arabidopsis.org/home/tair/Sequences/whole_chromosomes/
>>>>>>
>>>>>>
## Fastq source:
ftp://ftp-trace.ncbi.nih.gov/sra/sra-instant/reads/ByStudy/sra/SRP%2FSRP009%2FSRP009850/SRR390302/
>>>>>> buildindex(basename="tair10chr.fasta",
>>>>>> reference="tair10chr.fasta") align(index="tair10chr.fasta",
>>>>>> readfile1="SRR390302.fastq",
>>>>>> output_file="SRR390302_subread.sam", nthreads=8, indels=1,
>>>>>> TH1=2)
>>>>>>
>>>>>> ##################################### ## Process SAM file
>>>>>> with Rsamtools ## #####################################
>>>>>> library(Rsamtools) asBam(file="SRR390302_subread.sam",
>>>>>> destination="SRR390302_subread") [1]
>>>>>> "./data/SRR390302_subread.bam" reads<-
>>>>>> readBamGappedAlignments("SRR390302_subread.bam",
>>>>>> use.names=FALSE) Error in validObject(.Object) : invalid
>>>>>> class "GappedAlignments" object: 'ranges' contains values
>>>>>> outside of sequence bounds
>>>>>>
>>>>>> ## The error disappears if the following two reads are
>>>>>> removed from the SAM file: ## SRR390302.434558 and
>>>>>> SRR390302.2716043. Both reads have their end positions
>>>>>> outside ## the range of Chr5 as illustrated here:
>>>>>>
>>>>>> ## Relevant chunks of SAM file generated by Rsubread @SQ
>>>>>> SN:Chr1 LN:30427671 @SQ SN:Chr2 LN:19698289 @SQ
>>>>>> SN:Chr3 LN:23459830 @SQ SN:Chr4 LN:18585056 @SQ
>>>>>> SN:Chr5 LN:26975502 @SQ SN:ChrC LN:154478 @SQ
>>>>>> SN:ChrM LN:366924 SRR390302.1 0 Chr1 27018257
>>>>>> 2.0 35M * 0 0 TGG...ACC
>>>>>> III...GD)0 SRR390302.2 4 * 0 0
>>>>>> * * 0 0 TCC...AAA III...+I@
>>>>>> ... SRR390302.434558 0 Chr5 26975485
>>>>>> 5.0 35M * 0 0 ATG...TGG
>>>>>> III...&4( SRR390302.2716043 0 Chr5 26975486
>>>>>> 3.0 35M * 0 0 CAT...GGT
>>>>>> III...+2&
>>>>>>
>>>>>>
>>>>>>> sessionInfo()
>>>>>> R version 2.14.0 (2011-10-31) Platform:
>>>>>> x86_64-unknown-linux-gnu (64-bit)
>>>>>>
>>>>>> locale: [1] C
>>>>>>
>>>>>> attached base packages: [1] stats graphics grDevices
>>>>>> utils datasets methods base
>>>>>>
>>>>>> other attached packages: [1] Rsamtools_1.6.3
>>>>>> Biostrings_2.22.0 GenomicRanges_1.6.4 IRanges_1.12.5
>>>>>> Rsubread_1.4.2
>>>>>>
>>>>>> loaded via a namespace (and not attached): [1]
>>>>>> BSgenome_1.22.0 RCurl_1.8-0 XML_3.6-2
>>>>>> bitops_1.0-4.1 rtracklayer_1.14.4 tools_2.14.0
>>>>>> zlibbioc_1.0.0
>>>>>>
>>>>>> _______________________________________________
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>>>>>
>>>>>
>>>>>
>>>>>>
--
>>>>> Computational Biology Fred Hutchinson Cancer Research Center
>>>>> 1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109
>>>>>
>>>>> Location: M1-B861 Telephone: 206 667-2793
>>>>
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>>>
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>>>>
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