Too many % reads unmapped too short | 55.91%

[Sneh]

Hello,

             I have 75bp single end drosophila RNAseq reads. The FASTQC report the reads looks good. But when I try to align the reads to the reference dm3 genome, very little (39.16%) reads align to it.  I get a very high percentage of unmapped reads because they were too short.       

I tried multiple commands but nothing made much difference:

/data/administrative/Softwares/STAR-STAR_2.4.2a/bin/Linux_x86_64/STAR --genomeDir /data/administrative/Public_Databases/Reference/Drosophila/UCSC/Drosophila_melanogaster/UCSC/dm3/STAR_index/ --readFilesIn CD1.fastq

/data/administrative/Softwares/STAR-STAR_2.4.2a/bin/Linux_x86_64/STAR --genomeDir /data/administrative/Public_Databases/Reference/Drosophila/UCSC/Drosophila_melanogaster/UCSC/dm3/STAR_index/ --clip3pNbases 25 --sjdbOverhang 74 --readFilesIn CD1.fastq

Started job on |       May 03 12:44:09

                             Started mapping on |       May 03 12:49:20

                                    Finished on |       May 03 15:09:26

       Mapping speed, Million of reads per hour |       4.35

                          Number of input reads |       10156563

                      Average input read length |       75

                                    UNIQUE READS:

                   Uniquely mapped reads number |       3977709

                        Uniquely mapped reads % |       39.16%

                          Average mapped length |       72.43

                       Number of splices: Total |       104631

            Number of splices: Annotated (sjdb) |       0

                       Number of splices: GT/AG |       100227

                       Number of splices: GC/AG |       848

                       Number of splices: AT/AC |       299

               Number of splices: Non-canonical |       3257

                      Mismatch rate per base, % |       1.02%

                         Deletion rate per base |       0.02%

                        Deletion average length |       1.15

                        Insertion rate per base |       0.00%

                       Insertion average length |       1.87

                             MULTI-MAPPING READS:

        Number of reads mapped to multiple loci |       461916

             % of reads mapped to multiple loci |       4.55%

        Number of reads mapped to too many loci |       26928

             % of reads mapped to too many loci |       0.27%

                                  UNMAPPED READS:

       % of reads unmapped: too many mismatches |       0.00%

                 % of reads unmapped: too short |       55.91%

                     % of reads unmapped: other |       0.11%

I am not sure how to fix this issue. Any helpful advise is much appreciated. 

Thanks

Sneh

[Alexander Dobin]

Hi Sneh,

there are several reasons for high % of unmapped reads.

In your case, the sequencing quality scores are good - however, strangely, the mismatch error rate is quite high, 1%  - usually we get <0.5% from Illumina these days.

Two remaining suspects:

(i) too short sequencing insert - try to trim adapters

(ii) contamination with other species, adapters, etc - try to BLAT unmapped reads

Cheers

Alex

[Varun Gupta]

Hi,
With the help of Alex, I have tried using parameters

--outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 but then specify how much you want your read to be matched let's say 65bp(out of 75bp). So use option --outFilterMatchNmin 65.

Change these options to see at what level you get your % unmapped too short resolved.

Alex can you also explain what is actual meaning of mismatch rate per base. I have never given that parameter any thought while mapping.

Thanks

Varun

On Tuesday, May 10, 2016 at 12:08:27 PM UTC-4, Sneh wrote:

[Alexander Dobin]

Hi Varun,

the mismatch rate is calculated as follows: for all reads that were mapped, the number of mismatched bases is divided by the total number of mapped bases.

Cheers

Alex