# below 50 percents of reads are assigned in featurecount **URL:** \ **Category:** usegalaxy.org support\ **Created:** [June 1, 2021, 10:00am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068 "2021-06-01T10:00:43Z")\ **Posts on this page:** 16\ **Page:** 1
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 1, 2021, 10:00am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/1 "2021-06-01T10:00:43Z")
Hi. I am analyzing a human RNAseq single-end unstranded dataset. I have used the hg38 build-in index file in HISAT2 as a reference genome and the FeatureCounts build-in GTF file of hg38 as the GTF file in FeatureCounts. But only under 40% of reads are assigned in FeatureCounts. I Changed the GTF file many times and tried different GTF files but it was not effective. Now, what should I do to elevate the percentage of assignments? Is anything wrong with this dataset? Should not I use this dataset? ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 1, 2021, 10:13am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/2 "2021-06-01T10:13:36Z")
Welcome! Which tools are you using? ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 1, 2021, 10:28am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/3 "2021-06-01T10:28:52Z")
Hi. HISAT2 for mapping and featurecounts for annotation assignment. ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 1, 2021, 11:27am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/4 "2021-06-01T11:27:03Z")
Sorry, I mean before HISAT2; quality-checking/trimming? ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 1, 2021, 5:09pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/5 "2021-06-01T17:09:28Z")
FASTQC for quality checking and Cutadapt for trimming. ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 1, 2021, 6:00pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/6 "2021-06-01T18:00:57Z")
Ok. So, assuming you have high-quality reads, how was the HISAT2 alignment reports? Can you show it here? ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 6, 2021, 1:37pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/7 "2021-06-06T13:37:12Z")
Please accept my apologies for my delayed answer. For the multiQC report, I had to have summary reports of HISAT2 and I performed mapping by HISAT2 again to obtain summary files. Here is the MultiQC report: | Sample Name | % Aligned | | --- | --- | | HISAT2 on data 447\_ Mapping summary | 90.2% | | HISAT2 on data 449\_ Mapping summary | 86.9% | | HISAT2 on data 451\_ Mapping summary | 88.0% | | HISAT2 on data 453\_ Mapping summary | 86.7% | | HISAT2 on data 455\_ Mapping summary | 86.2% | | HISAT2 on data 457\_ Mapping summary | 85.9% | | HISAT2 on data 459\_ Mapping summary | 87.2% | | HISAT2 on data 461\_ Mapping summary | 88.0% | | HISAT2 on data 463\_ Mapping summary | 86.6% | | HISAT2 on data 465\_ Mapping summary | 85.0% | | HISAT2 on data 467\_ Mapping summary | 86.8% | | HISAT2 on data 469\_ Mapping summary | 86.0% | | HISAT2 on data 471\_ Mapping summary | 80.2% | | HISAT2 on data 473\_ Mapping summary | 81.6% | | HISAT2 on data 475\_ Mapping summary | 88.8% | | HISAT2 on data 477\_ Mapping summary | 89.9% | | HISAT2 on data 479\_ Mapping summary | 86.8% | | HISAT2 on data 481\_ Mapping summary | 85.5% | | HISAT2 on data 483\_ Mapping summary | 87.3% | | HISAT2 on data 485\_ Mapping summary | 83.0% | | HISAT2 on data 487\_ Mapping summary | 88.0% | | HISAT2 on data 489\_ Mapping summary | 87.1% | | HISAT2 on data 491\_ Mapping summary | 84.8% | | HISAT2 on data 493\_ Mapping summary | 85.8% | | HISAT2 on data 495\_ Mapping summary | 83.1% | | HISAT2 on data 497\_ Mapping summary | 87.9% | | HISAT2 on data 499\_ Mapping summary | 89.1% | | HISAT2 on data 501\_ Mapping summary | 86.7% | | HISAT2 on data 503\_ Mapping summary | 88.7% | | HISAT2 on data 505\_ Mapping summary | 88.8% | | HISAT2 on data 507\_ Mapping summary | 86.9% | | HISAT2 on data 509\_ Mapping summary | 85.1% | | HISAT2 on data 511\_ Mapping summary | 85.7% | | HISAT2 on data 513\_ Mapping summary | 87.7% | | HISAT2 on data 515\_ Mapping summary | 88.2% | | HISAT2 on data 517\_ Mapping summary | 87.3% | | HISAT2 on data 519\_ Mapping summary | 85.5% | | HISAT2 on data 521\_ Mapping summary | 87.0% | | HISAT2 on data 523\_ Mapping summary | 86.1% | | HISAT2 on data 525\_ Mapping summary | 88.8% | | HISAT2 on data 527\_ Mapping summary | 88.9% | | HISAT2 on data 529\_ Mapping summary | 84.6% | | HISAT2 on data 531\_ Mapping summary | 85.1% | | HISAT2 on data 533\_ Mapping summary | 83.8% | | HISAT2 on data 535\_ Mapping summary | 88.9% | | HISAT2 on data 537\_ Mapping summary | 88.4% | | HISAT2 on data 539\_ Mapping summary | 85.9% | | HISAT2 on data 541\_ Mapping summary | 88.0% | | HISAT2 on data 543\_ Mapping summary | 88.9% | | HISAT2 on data 545\_ Mapping summary | 88.7% | | HISAT2 on data 547\_ Mapping summary | 86.3% | | HISAT2 on data 549\_ Mapping summary | 87.2% | | HISAT2 on data 551\_ Mapping summary | 86.3% | | HISAT2 on data 553\_ Mapping summary | 85.6% | | HISAT2 on data 555\_ Mapping summary | 87.2% | | HISAT2 on data 557\_ Mapping summary | 90.4% | ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 6, 2021, 5:01pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/8 "2021-06-06T17:01:55Z")
And these are fastQC and Cutadapts reports prepaired by MultiQC. ![a](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/2/221500fc22ac0c4ed00f0327ca0a63cd7799baf5.jpeg) ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 6, 2021, 6:14pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/9 "2021-06-06T18:14:20Z")
@mmomeni, Mapping summary seems good, so I’d try check if your > [reads are mapped on regions that are not found in your annotation](https://www.biostars.org/p/388615/). Can you share what featurecounts’ parameters are you using? Maybe you can tweak some options related to stringency/overlap/quality, like the “`Allow reads to map to multiple features`”, `"Minimum mapping quality per read"`, `"Minimum fraction (of read) overlapping a feature`", etc… ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 7, 2021, 9:29am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/10 "2021-06-07T09:29:37Z")
No, I do not change any options you mentioned. All these parameters are set by default. ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 10, 2021, 11:14am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/11 "2021-06-10T11:14:39Z")
Hello again. So what do you think about the problem? Can I use the results of this RNAseq dataset? ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 10, 2021, 3:18pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/12 "2021-06-10T15:18:39Z")
Hello, @mmomeni. I’d still like to see your feedbacks about: > [@David](#): > > I’d try check if your > > > [reads are mapped on regions that are not found in your annotation](https://www.biostars.org/p/388615/). Have you read this? And > [@mmomeni](#): > > No, I do not change any options you mentioned. All these parameters are set by default. So you’re not worried about trying to refine for (possible) better parameters? ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 11, 2021, 6:24am UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/13 "2021-06-11T06:24:00Z")
Yes, I have read that but it did not help me because the cause of un assignment, in that case, was NoFeature and Ambiguity. As you see below the most unassigned reads are because of multi mapping: ![featureCounts_assignment_plot](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/3/36d5a92374b4f3a42a073ee4770b9009bf2c7f41.png) By considering this which of the mentioned parameters should be changed? ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 11, 2021, 2:08pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/14 "2021-06-11T14:08:50Z")
Now that’s a good report. > [@David](#): > > Allow reads to map to multiple features Have you tried this? ---
**Author:** ![mmomeni](https://avatars.discourse-cdn.com/v4/letter/m/a587f6/32.png) [@mmomeni](https://help.galaxyproject.org/u/mmomeni)\ **Post date:** [June 13, 2021, 12:12pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/15 "2021-06-13T12:12:53Z")
Now I tried it and the percentages of reads assigned became a little better(38-60 percent). But something strange happened the percentage of Unassigned\_NoFeatures increased a lot! What is the reason for that? ![featureCounts2_assignment_plot](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/d/de213434c6143f3487974e303c42009f3066ef0b.png) ---
**Author:** ![David](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/david/32/7031_2.png) [@David](https://help.galaxyproject.org/u/David)\ **Post date:** [June 13, 2021, 2:08pm UTC](https://help.galaxyproject.org/t/below-50-percents-of-reads-are-assigned-in-featurecount/6068/16 "2021-06-13T14:08:41Z")
I guess can’t give you any better help, Have you compared all the options from `Allow reads to map to multiple features`?