[BioC] Rsubread/Rsamtools mappings outside of chromosome ranges
Wei Shi
shi at wehi.EDU.AU
Tue Feb 14 03:18:38 CET 2012
Hi Thomas,
Although subread can map junction reads to the genome which is enough for performing an RNA-seq expression analysis, it does not have the capacity to identify splicing points in the genome. However, the subjunc function included in Rsubread package can be used for this purpose. It takes as input the SAM file outputted from the align() function and then uses the discovered junction reads (CIGAR strings of such reads contain letter 'N') to find exon-exon junctions. It can detect junction locations in the reads which are as close as 4bp from the ends of the reads.
We have compared subjunc with tophat, mapsplice and splicemap and obtained quite nice results. I could not make these results publicly available before we get our algorithms published, but I will be happy to send you some of the evaluation results off the list.
Cheers,
Wei
The subjunc function in Rsubread package
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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