PMID- 32061258
OWN - NLM
STAT- MEDLINE
DCOM- 20210108
LR  - 20210110
IS  - 2049-2618 (Electronic)
IS  - 2049-2618 (Linking)
VI  - 8
IP  - 1
DP  - 2020 Feb 15
TI  - A pipeline for targeted metagenomics of environmental bacteria.
PG  - 21
LID - 10.1186/s40168-020-0790-7 [doi]
LID - 21
AB  - BACKGROUND: Metagenomics and single cell genomics provide a window into the 
      genetic repertoire of yet uncultivated microorganisms, but both methods are 
      usually taxonomically untargeted. The combination of fluorescence in situ 
      hybridization (FISH) and fluorescence activated cell sorting (FACS) has the 
      potential to enrich taxonomically well-defined clades for genomic analyses. 
      METHODS: Cells hybridized with a taxon-specific FISH probe are enriched based on 
      their fluorescence signal via flow cytometric cell sorting. A recently developed 
      FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high 
      signal intensities required for flow cytometric sorting while maintaining the 
      integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are 
      subjected to shotgun sequencing, resulting in targeted metagenomes of low 
      diversity. RESULTS: Pure cultures of different taxonomic groups were used to (1) 
      adapt and optimize the HCR-FISH protocol and (2) assess the effects of various 
      cell fixation methods on both the signal intensity for cell sorting and the 
      quality of subsequent genome amplification and sequencing. Best results were 
      obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and 
      genome assembly quality. Our newly developed pipeline was successfully applied to 
      a marine plankton sample from the North Sea yielding good quality metagenome 
      assembled genomes from a yet uncultivated flavobacterial clade. CONCLUSIONS: With 
      the developed pipeline, targeted metagenomes at various taxonomic levels can be 
      efficiently retrieved from environmental samples. The resulting metagenome 
      assembled genomes allow for the description of yet uncharacterized microbial 
      clades. Video abstract.
FAU - Grieb, Anissa
AU  - Grieb A
AD  - Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359, Bremen, 
      Germany.
FAU - Bowers, Robert M
AU  - Bowers RM
AD  - DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Mail Stop: 
      91R183, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
FAU - Oggerin, Monike
AU  - Oggerin M
AD  - Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359, Bremen, 
      Germany.
FAU - Goudeau, Danielle
AU  - Goudeau D
AD  - DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Mail Stop: 
      91R183, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
FAU - Lee, Janey
AU  - Lee J
AD  - DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Mail Stop: 
      91R183, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
FAU - Malmstrom, Rex R
AU  - Malmstrom RR
AD  - DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Mail Stop: 
      91R183, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
FAU - Woyke, Tanja
AU  - Woyke T
AD  - DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Mail Stop: 
      91R183, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
FAU - Fuchs, Bernhard M
AU  - Fuchs BM
AUID- ORCID: 0000-0001-9828-1290
AD  - Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359, Bremen, 
      Germany. bfuchs@mpi-bremen.de.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Video-Audio Media
DEP - 20200215
PL  - England
TA  - Microbiome
JT  - Microbiome
JID - 101615147
RN  - 0 (DNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - Bacteria/*classification/*genetics/isolation & purification
MH  - DNA, Bacterial/genetics
MH  - *Environmental Microbiology
MH  - Flow Cytometry
MH  - In Situ Hybridization, Fluorescence
MH  - Metagenome
MH  - Metagenomics/*methods
MH  - RNA, Ribosomal, 16S/genetics
MH  - *Water Microbiology
PMC - PMC7024552
OTO - NOTNLM
OT  - Cell fixation
OT  - FACS
OT  - HCR-FISH
OT  - Mini-metagenomics
COIS- The authors declare that they have no competing interests.
EDAT- 2020/02/18 06:00
MHDA- 2021/01/09 06:00
PMCR- 2020/02/15
CRDT- 2020/02/17 06:00
PHST- 2019/08/23 00:00 [received]
PHST- 2020/01/19 00:00 [accepted]
PHST- 2020/02/17 06:00 [entrez]
PHST- 2020/02/18 06:00 [pubmed]
PHST- 2021/01/09 06:00 [medline]
PHST- 2020/02/15 00:00 [pmc-release]
AID - 10.1186/s40168-020-0790-7 [pii]
AID - 790 [pii]
AID - 10.1186/s40168-020-0790-7 [doi]
PST - epublish
SO  - Microbiome. 2020 Feb 15;8(1):21. doi: 10.1186/s40168-020-0790-7.