# High proportion of Unmapped reads in metagenomic data **URL:** \ **Category:** HUMAnN\ **Created:** [October 19, 2023, 1:19am UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095 "2023-10-19T01:19:57Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![mhassan](https://avatars.discourse-cdn.com/v4/letter/m/6f9a4e/32.png) [@mhassan](https://forum.biobakery.org/u/mhassan)\ **Post date:** [October 19, 2023, 1:19am UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/1 "2023-10-19T01:19:57Z")
Hello, I am working with lake water sample. My focus is on harmful cyanobacteria. I ran metagenomic sequence using HUMAnN3. **Configuration I used for my sample is :** HUMAnN Configuration ( Section : Name = Value ) database\_folders : nucleotide = /home/hassan/Desktop/hdatabases/chocophlan database\_folders : protein = /home/hassan/Desktop/hdatabases/uniref50ecf database\_folders : utility\_mapping = /home/hassan/Desktop/b/utility\_mapping run\_modes : resume = False run\_modes : verbose = False run\_modes : bypass\_prescreen = False run\_modes : bypass\_nucleotide\_index = False run\_modes : bypass\_nucleotide\_search = False run\_modes : bypass\_translated\_search = False run\_modes : threads = 1 alignment\_settings : evalue\_threshold = 1.0 alignment\_settings : prescreen\_threshold = 0.01 alignment\_settings : translated\_subject\_coverage\_threshold = 50.0 alignment\_settings : translated\_query\_coverage\_threshold = 90.0 alignment\_settings : nucleotide\_subject\_coverage\_threshold = 50.0 alignment\_settings : nucleotide\_query\_coverage\_threshold = 90.0 output\_format : output\_max\_decimals = 10 output\_format : remove\_stratified\_output = False output\_format : remove\_column\_description\_output = False **Outputs I got:** Removing spaces from identifiers in input file … Running metaphlan … Found g\_\_GGB43952.s\_\_GGB43952\_SGB61317 : 24.13% of mapped reads ( ) Found g\_\_GGB43067.s\_\_GGB43067\_SGB57480 : 13.42% of mapped reads ( ) Found g\_\_GGB46342.s\_\_GGB46342\_SGB64120 : 13.10% of mapped reads ( ) Found g\_\_GGB24856.s\_\_GGB24856\_SGB81948 : 8.95% of mapped reads ( ) Found g\_\_GGB43951.s\_\_GGB43951\_SGB61315 : 6.47% of mapped reads ( ) Found g\_\_GGB35689.s\_\_GGB35689\_SGB85076 : 6.18% of mapped reads ( ) Found g\_\_Polynucleobacter.s\_\_Polynucleobacter\_sp\_MWH\_UH24A : 4.19% of mapped reads ( ) Found g\_\_GGB25977.s\_\_GGB25977\_SGB37971 : 3.43% of mapped reads ( ) Found g\_\_GGB59202.s\_\_GGB59202\_SGB80969 : 3.40% of mapped reads ( ) Found t\_\_SGB13449 : 3.15% of mapped reads ( s\_\_Cylindrospermopsis\_raciborskii,g\_\_Raphidiopsis.s\_\_Raphidiopsis\_brookii ) Found t\_\_SGB24761 : 2.51% of mapped reads ( s\_\_Cuspidothrix\_issatschenkoi ) Found t\_\_SGB24471 : 2.42% of mapped reads ( s\_\_Phenylobacterium\_sp\_HYN0004 ) Found g\_\_GGB43953.s\_\_GGB43953\_SGB61318 : 2.39% of mapped reads ( ) Found g\_\_Candidatus\_Methylopumilus.s\_\_Candidatus\_Methylopumilus\_rimovensis : 1.20% of mapped reads ( ) Found g\_\_GGB57651.s\_\_GGB57651\_SGB79249 : 0.84% of mapped reads ( ) Found g\_\_GGB34754.s\_\_GGB34754\_SGB82226 : 0.65% of mapped reads ( ) Found t\_\_SGB28829 : 0.58% of mapped reads ( s\_\_Pelagibacterales\_bacterium ) Found g\_\_Pseudanabaena.s\_\_Pseudanabaena\_sp\_FACHB\_1050 : 0.54% of mapped reads ( ) Found t\_\_SGB24760 : 0.36% of mapped reads ( s\_\_Anabaena\_sp\_CRKS33,g\_\_Dolichospermum.s\_\_Dolichospermum\_planctonicum,g\_\_Dolichospermum.s\_\_Dolichospermum\_flos\_aquae,g\_\_Dolichospermum.s\_\_Dolichospermum\_sp\_FACHB\_1091,g\_\_Anabaena.s\_\_Anabaena\_sp\_FACHB\_1250,g\_\_Anabaena.s\_\_Anabaena\_sp\_FACHB\_1391 ) Found t\_\_SGB13518 : 0.28% of mapped reads ( s\_\_Microcystis\_aeruginosa,g\_\_Microcystis.s\_\_Microcystis\_viridis,g\_\_Microcystis.s\_\_Microcystis\_wesenbergii,g\_\_Microcystis.s\_\_Microcystis\_sp\_0824,g\_\_Microcystis.s\_\_Microcystis\_sp\_T1\_4,g\_\_Microcystis.s\_\_Microcystis\_sp\_LEGE\_00066,g\_\_Microcystis.s\_\_Microcystis\_sp\_MC19,g\_\_Microcystis.s\_\_Microcystis\_sp\_LEGE\_08355,g\_\_Microcystis.s\_\_Microcystis\_flos\_aquae ) Found g\_\_Alphaproteobacteria\_unclassified.s\_\_alpha\_proteobacterium\_SCGC\_AAA028\_D10 : 0.28% of mapped reads ( g\_\_Alphaproteobacteria\_unclassified.s\_\_alpha\_proteobacterium\_SCGC\_AAA027\_C06,g\_\_Alphaproteobacteria\_unclassified.s\_\_alpha\_proteobacterium\_SCGC\_AAA027\_L15 ) Found g\_\_GGB73741.s\_\_GGB73741\_SGB49722 : 0.27% of mapped reads ( ) Found g\_\_GGB32003.s\_\_GGB32003\_SGB45716 : 0.16% of mapped reads ( ) Found g\_\_GGB43055.s\_\_GGB43055\_SGB60296 : 0.14% of mapped reads ( ) Found g\_\_GGB24725.s\_\_GGB24725\_SGB36612 : 0.13% of mapped reads ( ) Found g\_\_Limnohabitans.s\_\_Limnohabitans\_sp\_103DPR2 : 0.11% of mapped reads ( g\_\_Limnohabitans.s\_\_Limnohabitans\_sp\_Hippo4 ) Found g\_\_Actinomycetia\_unclassified.s\_\_actinobacterium\_SCGC\_AAA028\_A23 : 0.10% of mapped reads ( ) Found g\_\_Pseudanabaena.s\_\_Pseudanabaena\_yagii : 0.10% of mapped reads ( ) Found t\_\_SGB5711 : 0.09% of mapped reads ( s\_\_Candidatus\_Nanopelagicus\_limnes ) Found t\_\_SGB13423 : 0.08% of mapped reads ( s\_\_Planktothrix\_agardhii,g\_\_Planktothrix.s\_\_Planktothrix\_rubescens,g\_\_Planktothrix.s\_\_Planktothrix\_prolifica ) Found g\_\_GGB56956.s\_\_GGB56956\_SGB78416 : 0.08% of mapped reads ( ) Found g\_\_GGB32489.s\_\_GGB32489\_SGB48813 : 0.08% of mapped reads ( ) Found g\_\_GGB62809.s\_\_GGB62809\_SGB85028 : 0.07% of mapped reads ( ) Found t\_\_SGB24763 : 0.06% of mapped reads ( s\_\_Sphaerospermopsis\_kisseleviana,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_kisseleviana,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_sp\_FACHB\_1194,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_sp\_LEGE\_08334,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_sp\_FACHB\_1094,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_reniformis,g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_sp\_LEGE\_00249 ) Found g\_\_GGB46492.s\_\_GGB46492\_SGB64353 : 0.02% of mapped reads ( ) Found g\_\_GGB43954.s\_\_GGB43954\_SGB61319 : 0.02% of mapped reads ( ) Found g\_\_GGB44382.s\_\_GGB44382\_SGB61797 : 0.01% of mapped reads ( ) **Total species selected from prescreen: 71** **Selected species explain 99.99% of predicted community composition** Creating custom ChocoPhlAn database … Running bowtie2-build … Running bowtie2 … **Total bugs from nucleotide alignment: 13** g\_\_Cuspidothrix.s\_\_Cuspidothrix\_issatschenkoi: 46989 hits g\_\_Anabaena.s\_\_Anabaena\_sp\_CRKS33: 10985 hits g\_\_Pelagibacterales\_unclassified.s\_\_Pelagibacterales\_bacterium: 6402 hits g\_\_Cylindrospermopsis.s\_\_Cylindrospermopsis\_raciborskii: 30055 hits g\_\_Phenylobacterium.s\_\_Phenylobacterium\_sp\_HYN0004: 28182 hits g\_\_Microcystis.s\_\_Microcystis\_aeruginosa: 12437 hits g\_\_Raphidiopsis.s\_\_Raphidiopsis\_brookii: 47806 hits g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_kisseleviana: 3140 hits g\_\_Candidatus\_Nanopelagicus.s\_\_Candidatus\_Nanopelagicus\_limnes: 7823 hits g\_\_Planktothrix.s\_\_Planktothrix\_agardhii: 1498 hits g\_\_Microcystis.s\_\_Microcystis\_flos\_aquae: 1067 hits g\_\_Microcystis.s\_\_Microcystis\_wesenbergii: 661 hits g\_\_Planktothrix.s\_\_Planktothrix\_rubescens: 835 hits **Total gene families from nucleotide alignment: 7904** **Unaligned reads after nucleotide alignment: 99.2391962932 %** Running diamond … Aligning to reference database: uniref50\_201901b\_ec\_filtered.dmnd **Total bugs after translated alignment: 14** g\_\_Cuspidothrix.s\_\_Cuspidothrix\_issatschenkoi: 46989 hits g\_\_Anabaena.s\_\_Anabaena\_sp\_CRKS33: 10985 hits g\_\_Pelagibacterales\_unclassified.s\_\_Pelagibacterales\_bacterium: 6402 hits g\_\_Cylindrospermopsis.s\_\_Cylindrospermopsis\_raciborskii: 30055 hits g\_\_Phenylobacterium.s\_\_Phenylobacterium\_sp\_HYN0004: 28182 hits g\_\_Microcystis.s\_\_Microcystis\_aeruginosa: 12437 hits g\_\_Raphidiopsis.s\_\_Raphidiopsis\_brookii: 47806 hits g\_\_Sphaerospermopsis.s\_\_Sphaerospermopsis\_kisseleviana: 3140 hits g\_\_Candidatus\_Nanopelagicus.s\_\_Candidatus\_Nanopelagicus\_limnes: 7823 hits g\_\_Planktothrix.s\_\_Planktothrix\_agardhii: 1498 hits g\_\_Microcystis.s\_\_Microcystis\_flos\_aquae: 1067 hits g\_\_Microcystis.s\_\_Microcystis\_wesenbergii: 661 hits g\_\_Planktothrix.s\_\_Planktothrix\_rubescens: 835 hits **unclassified: 1965349 hits** **Total gene families after translated alignment: 55375** **Unaligned reads after translated alignment: 92.6032063024 %** After running metaphlan 71 spices were identified but most of them (58) are unclassified species. 13 species were classified in metaphlan which is consistent with bowtie2 and diamond (uniref50ecfiltered). These species are mostly related to algal bloom which is also my interest as well. But my questions are: 1. Is there any way to increase the number of identified species, decease % of unaligned reads of nucleotide and translated alignment (which is currently approx. 99% and 92% respectively)? 2. What could be the reason for finding only algal bloom related species in my sample although a major portion is unaligned ? 3. what is the difference of ec-filtered database and not ec-filtered database? 4. If there is no way to increase % aligned reads do you think this % is usual for these kind of water sample? ( I have also attached the log file of my run for better understanding) [run0043\_lane9\_read2\_indexN726-S518=ENN-8-17-16.txt|attachment](https://forum.biobakery.org/uploads/short-url/giGmWcSDEQj8Cczk7h49IsIgU4G.txt) (113.8 KB) Thanks, Hassan ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [October 20, 2023, 8:31pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/2 "2023-10-20T20:31:22Z")
1. The bottom line (according to your MetaPhlAn output) is that most of the species in these samples are just not very well characterized. This means that they are missing pangenomes for alignment in HUMAnN 3. HUMAnN 4 will improve this by including pangenomes populated by MAGs from different environments, but we are always playing “catchup” to some extent on the less-well studied environments. For translated search, are you using the EC filtered database (per your question 3)? That only contains proteins that have an EC annotation (~10%), so mapping to that database will produce fewer translated hits, but is faster and more interpretable. 2. I am not an expert on the biology here, but perhaps the species responsible for algal blooms have been better studied / sequenced than the background species? This is true for human pathogens (as an analogy). 3. See comment in 1. 4. I have not worked with this sort of sample enough to have a good % mapping rate in mind. You might check the forum for rates reported by other HUMAnN users with similar environmental communities for comparison? ---
**Author:** ![mhassan12](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mhassan12/32/2451_2.png) [@mhassan12](https://forum.biobakery.org/u/mhassan12)\ **Post date:** [October 23, 2023, 7:53pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/3 "2023-10-23T19:53:05Z")
Thanks a lot for your reply. It helped me a lot. Currently I am facing a issue with metaphlan bug list. Log file of the run: [062615a\_S9\_L001\_R1\_001.txt](https://forum.biobakery.org/uploads/short-url/XsA6kG3K0Bm1fqI3gnKauF2t4G.txt) (117.3 KB) **Here is the concerning portion of my output:** Creating output directory: /home/hassan/Desktop/bouy/withoutbypass Output files will be written to: /home/hassan/Desktop/bouy/withoutbypass Decompressing gzipped file … Removing spaces from identifiers in input file … Running metaphlan … Found t\_\_SGB24760 : 100.00% of mapped reads ( s\_\_Anabaena\_sp\_CRKS33,g\_\_Dolichospermum.s\_\_Dolichospermum\_planctonicum,g\_\_Dolichospermum.s\_\_Dolichospermum\_flos\_aquae,g\_\_Dolichospermum.s\_\_Dolichospermum\_sp\_FACHB\_1091,g\_\_Anabaena.s\_\_Anabaena\_sp\_FACHB\_1250,g\_\_Anabaena.s\_\_Anabaena\_sp\_FACHB\_1391 ) Total species selected from prescreen: 7 [062615a\_S9\_L001\_R1\_001\_metaphlan\_bugs\_list.tsv](https://forum.biobakery.org/uploads/short-url/aQvpQ64QkD7mNOUbbSK6toC6tNB.tsv) (2.1 KB) Although total species selected from prescreen is 7. In the metaphlan bug list file has only 1 species which is below: k\_\_Bacteria|p\_\_Cyanobacteria|c\_\_Cyanobacteria\_unclassified|o\_\_Nostocales|f\_\_Aphanizomenonaceae|g\_\_Dolichospermum|s\_\_Dolichospermum\_circinale What could be the reason for this? How can I get the complete bug list with their relative abundance? Thanks ---
**Author:** ![mhassan](https://avatars.discourse-cdn.com/v4/letter/m/6f9a4e/32.png) [@mhassan](https://forum.biobakery.org/u/mhassan)\ **Post date:** [October 25, 2023, 3:02pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/4 "2023-10-25T15:02:31Z")
Hi @franzosa , I would appreciate your kind suggestion regarding this issue. Thanks ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [October 26, 2023, 8:12pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/5 "2023-10-26T20:12:56Z")
This is a function of the compatibility mode between HUMAnN 3 and MetaPhlAn 4. MetaPhlAn 4 is reporting an SGB that was historically grouped into multiple named species, so HUMAnN 3 will map against all of those species pangenomes pangenomes. You can find the full taxonomic profile under the HUMAnN temp folder for your sample. ---
**Author:** ![mhassan12](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mhassan12/32/2451_2.png) [@mhassan12](https://forum.biobakery.org/u/mhassan12)\ **Post date:** [October 27, 2023, 3:18pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/6 "2023-10-27T15:18:57Z")
![Screenshot from 2023-10-27 11-12-25](https://canada1.discourse-cdn.com/flex027/uploads/biobakery/original/2X/0/0a7c85f002d1a91e6784df583a06b8f8cd9eefcf.png) ![Screenshot from 2023-10-27 11-13-05](https://canada1.discourse-cdn.com/flex027/uploads/biobakery/original/2X/f/fa8389332be5c51133721706bcb6ddb546b74e6e.png) Hi @franzosa , There are the output from HUMAnN. Can you please tell me which file contains full taxonomic profile? [062615a\_S9\_L001\_R1\_001\_metaphlan\_bugs\_list.tsv](https://forum.biobakery.org/uploads/short-url/aQvpQ64QkD7mNOUbbSK6toC6tNB.tsv) (2.1 KB) This file contains k\_\_Bacteria|p\_\_Cyanobacteria|c\_\_Cyanobacteria\_unclassified|o\_\_Nostocales|f\_\_Aphanizomenonaceae|g\_\_Dolichospermum|s\_\_Dolichospermum\_circinale on the other hand, [062615a\_S9\_L001\_R1\_001\_pathabundance.tsv](https://forum.biobakery.org/uploads/short-url/c2PGyVQ42mPcUH1JD9pxQsUnHqM.tsv) (1.2 KB) this file contains: g\_\_Anabaena.s\_\_Anabaena\_sp\_CRKS33 as stratified output. Which are not same. Can you please tell me why is these difference? Thanks ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [November 10, 2023, 9:47pm UTC](https://forum.biobakery.org/t/high-proportion-of-unmapped-reads-in-metagenomic-data/6095/7 "2023-11-10T21:47:55Z")
I think my above response addresses this?