Analysis of en­vir­on­mental mi­crobes with the help of phylo­Flash

Researchers are developing a user-friendly method to reconstruct and analyze SSU rRNA from raw metagenome data

November 09, 2020

Mi­cro­bi­o­lo­gists tra­di­tion­ally de­term­ine which or­gan­isms they are deal­ing with us­ing the small sub­unit ri­bosomal RNA or in short SSU rRNA gene. This marker gene al­lows to identify al­most any liv­ing creature, be it a bac­terium or an an­imal, and thus as­sign it to its place in the tree of life. Re­search­ers at the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy in Bre­men now present a method that closes this gap and makes it pos­sible to re­con­struct and ana­lyze SSU rRNA from raw meta­gen­ome data.

Once the po­s­i­tion in the tree of life is known, spe­cific DNA probes can be de­signed to make the or­gan­isms vis­ible in an ap­proach called FISH (fluor­es­cence in situ hy­brid­iz­a­tion). FISH has many ap­plic­a­tions, for ex­ample to sort cells, or to mi­cro­scop­ic­ally re­cord their mor­pho­logy or spa­tial po­s­i­tion. This ap­proach – which leads from DNA to gene to tree and probe to im­age – is called the “full-cycle rRNA ap­proach”. To make the SSU rRNA meas­ur­able, it is usu­ally amp­li­fied with poly­merase chain re­ac­tion (PCR).

Today, however, PCR is in­creas­ingly be­ing re­placed by so-called meta­ge­n­om­ics, which re­cord the en­tirety of all genes in a hab­itat. Rapid meth­od­o­lo­gical ad­vances now al­low the fast and ef­fi­cient pro­duc­tion of large amounts of such meta­ge­n­omic data. The ana­lysis is per­formed us­ing sig­ni­fic­antly shorter DNA se­quence seg­ments – much shorter than the SSU gene – which are then la­bor­i­ously as­sembled and placed into so-called meta­ge­n­om­ic­ally as­sembled gen­omes (MAGs). The short gene snip­pets do not provide com­plete SSU rRNA, and even in many as­sem­blies and MAGs we do not find this im­port­ant marker gene. This makes it hard to mo­lecu­larly identify or­gan­isms in meta­gen­omes, to com­pare them to ex­ist­ing data­bases or even to visu­al­ize them spe­cific­ally with FISH.

phylo­Flash provides rem­edy

“This soft­ware called phyloFlash, which is freely available through GitHub, com­bines the full-cycle rRNA ap­proach for iden­ti­fic­a­tion and visu­al­iz­a­tion of non-cul­tiv­ated mi­croor­gan­isms with meta­ge­n­omic ana­lysis; both tech­niques are well es­tab­lished at the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy in Bre­men,” ex­plains Har­ald Gruber-Vodicka, who chiefly de­veloped the method.

“phylo­Flash com­prises all ne­ces­sary steps, from the pre­par­a­tion of the ne­ces­sary gen­ome data­base (in this case SILVA), data ex­trac­tion and taxo­nomic clas­si­fic­a­tion, through as­sembly, to the link­ing of SSU rRNA se­quences and MAGs”. In ad­di­tion, the soft­ware is very user-friendly and both in­stall­a­tion and ap­plic­a­tion are largely auto­mated.

Es­pe­cially suit­able for simple com­munit­ies

The two lead authors share a passion for bioinformatics and symbiotic critters in marine sands. Here, they are searching for small worms under the microscope at Carrie Bow Cay Field Station in Belize.

Gruber-Vodicka and his col­league Brandon Seah – who are shared first au­thors of the pub­lic­a­tion now present­ing phylo­Flash in the journal mSystems – come from sym­bi­osis re­search. The com­munit­ies they are deal­ing with in this field of re­search are com­par­at­ively simple: Usu­ally a host or­gan­ism lives to­gether with one or a hand­ful of mi­cro­bial sym­bionts. Such com­munit­ies are par­tic­u­larly well suited for ana­lysis with phylo­Flash. “For ex­ample, we do a lot of re­search on the deep-sea mus­sel Bathymodiolus, which is home to sev­eral bac­terial sub­ten­ants,” says Gruber-Vodicka. “With the help of this well-stud­ied com­munity, we were able to test whether and how re­li­ably phylo­Flash works”. And in­deed, the new soft­ware re­li­ably iden­ti­fied both the mus­sel and its vari­ous sym­bionts. Niko Leisch, also a sym­bi­osis re­searcher at the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy, tested phylo­Flash on small mar­ine round­worms. Ana­lyses of vari­ous such nem­at­odes showed that some of the spe­cies of these in­con­spicu­ous worms might be as­so­ci­ated with sym­bionts. “These ex­cit­ing glimpses un­der­line the great po­ten­tial of our simple and fast method”, Gruber-Vodicka points out.

Open­Source and all-pur­pose

phylo­Flash is an Open­Source soft­ware. Ex­tens­ive doc­u­ment­a­tion and a very act­ive com­munity en­sure its con­tinu­ous test­ing and fur­ther de­vel­op­ment. “phylo­Flash is cer­tainly not only in­ter­est­ing for mi­cro­bi­o­lo­gists,” em­phas­izes Gruber-Vodicka. “Already now, nu­mer­ous sci­ent­ists from di­verse fields of re­search make use of our soft­ware. The simple in­stall­a­tion was cer­tainly help­ful in this re­spect, as it lowers the threshold for use”. This easy ac­cess and in­ter­act­ive char­ac­ter is also par­tic­u­larly im­port­ant to Brandon Seah, who now works at the Max Planck In­sti­tute for De­vel­op­mental Bio­logy: “The most sat­is­fy­ing thing for me about this pro­ject is to see other people us­ing our soft­ware to drive their own re­search for­ward,” says Seah. "From the be­gin­ning, we've ad­ded fea­tures and de­veloped the soft­ware in re­sponse to user feed­back. These users are not just col­leagues down the hall, but also people from the other side of the world who have given it a try and got­ten in touch with us on­line. It un­der­lines how open-source is more pro­duct­ive and be­ne­fi­cial both for soft­ware de­vel­op­ment and for sci­ence.” 

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