PROBIOTIC GENOMES: SEQUENCING AND ANNOTATION IN THE PAST DECADEHTML Full Text
PROBIOTIC GENOMES: SEQUENCING AND ANNOTATION IN THE PAST DECADE
Joel P. Joseph
Department of Genetic Engineering, SRM University, Kattankulathur - 603203, Tamil Nadu, India.
ABSTRACT: Probiotics are live microorganisms that confer many health benefits to the host when administered in adequate quantities. These health benefits have garnered much attention towards Probiotics and have given an impetus to their use as dietary supplements for the improvement of general health and as adjuvant therapies for certain diseases. The increased demand for probiotic products in the recent times has provided the thrust for probiotic research applied to several areas of human biology. The advances in genomic technologies have further facilitated the sequencing of the genomes of such probiotic bacteria and their genomic analyses to identify the genes that endow the beneficial effects they are known to exert. This work reviews the application of genomic strategies on probiotic bacteria, while providing the details about the probiotic strains whose genome sequences are available. It also consolidates the Genomic tools used for the sequencing, assembly and annotation of the probiotic genes and how it has helped in comparative genomic analyses.
Probiotics, Genome, Bacteria, Disease
INTRODUCTION: Probiotics can be defined as live non-pathogenic microorganisms that present health benefits to the host when administered in adequate quantities 1, 2. They fall under the class of functional foods 3, 4, and their health benefits encompass multiple facets of human health including improvement of intestinal health through the regulation of gut microflora 4 - 6, prevention of enteric, respiratory tract, and urogenital infections 2, 4, 7, 8, stimulation of immune system 9, anti-allergic activity 2, anti-cancer effects 4, 7, anti-microbial effects 10 - 12, and cholesterol-lowering effects 13 - 16. The growth of the global probiotic market has in turn served as an impetus to probiotic research driving the adoption of modern scientific technologies in studying the genetics and biology of probiotic microorganisms 3.
The term Probiotic finds its origin from the Greek words pro meaning for and biotikos meaning pertaining to life 17. With the earliest clues about the involvement of probiotics in health benefits dating back to the biblical times and the times of the ancient civilizations like the Roman empire, the history of probiotics go way back in time 18. The identification and isolation of gut microflora eventually paved way to the isolation of probiotic species and the study of their health benefits 4, 5, 17, 19, 20. Among the bacterial species that fall under the spectrum of probiotics are the non-pathogenic species within the genera of Lactobacillus, Bifidobacterium, Clostridium, Bacillus, Escherichia, and Enterococcus 17. However, since several years, species within the genera of Lactobacillus and Bifidobacterium have dominated probiotic market21.
In the late 1990s and the early 2000, advances in sequencing technologies facilitated whole genome sequencing of several bacterial pathogens including Mycobaterium tuberculosis, Pseudomons aeroginosa, and enterohaemorrhagic Escherichia coli 22 - 24.
In the recent past, however, the demand for probiotics has served as an impetus for the application of sequencing strategies and genomic technologies to obtain and analyze the whole genome sequences of several probiotic bacteria 25-27. Thus, advances in genomic technologies and computational strategies have facilitated the characterization of microbial population, particularly probiotic bacteria 28. The forthcoming section of this review articulates predominant probiotic species whose whole genome or draft genome sequences have been made available in public databases.
Genomic Technologies in Probiotic Research: One of the earliest whole genome sequencing projects of a probiotic species (Lactobacillus johnsonii NCC 533) was published as early as 2004 27. The sequencing strategy that was used for this project was the whole genome shotgun sequencing technology 27. There on, several sequencing projects of probiotic genomes were published in the years that followed, with a rise in the number of such projects in the very recent years 25, 29 - 32. Furthermore, there has been a gradual change in the sequencing technologies adopted overtime for such projects thus facilitating more genomes to be sequenced, assembled and annotated in shorter durations of time 25, 29 - 32.
While initial genome sequencing strategies embraced the traditional Sanger sequencing methods 25, more advanced sequencing technologies that are collectively referred to as the Next Generation Sequencing (NGS) technologies have been eventually adopted 26, 33- 35. The genome sequencing of probiotic species until or before 2010 was accomplished by the traditional Sanger sequencing method and shotgun sequencing technique. These include the genome sequencing of Lactobacillus johnsonii NCC 533 and Bifidobacterium animalis subsp. lactis AD01125, 27. Post 2010, NGS technologies have been adopted for genome sequencing with the prominent ones being 454 pyrosequencing technology, Illumina/ Solexa paired end sequencing technology, Ion Torrent sequencing technology, and Pacific BioSciences sequencing technology 26, 33-35.
In 2011, most of the probiotic genomes were sequenced using Roche 454 GS FLX pyrosequencer. These include the genome sequences of Lactobacillus amylovorus 33, 36, Lactobacillus ruminis37, Lactobacillus coryniformis 38, Lactobacillus animalis 39, Lactobacillus cypricasei 40, Lactobacillus sanfranciscensis 41, and Lactobacillus kefiranofaciens 31 among others. In some cases, a combination of two different sequencing technologies has been adopted. For instance, in case of Lactobacillus sanfranciscensis genome sequencing, a combination of Roche 454 GS FLX pyrosequencing technology and Sanger sequencing was adopted 41. Similarly, in case of Lactobacillus kefiranofaciens, Roche 454 GS FLX pyrosequencing technology was combined with Illumina Genome Analyzer IIx Solexa high throughput sequencing technology to sequence the genome 31.
In most cases, genome assembly was done using different versions of Newbler assembler 33, 39 except in a few cases where gsAssembler 36, 37 or CLC Genomics Workbench 37 Phred-Phrap-Consed software package 41. Genome annotation was done using the Rapid Assembly using Subsystems Technology (RAST) server 38 - 40, often combined with Glimmer 38, tRNAscan-SE 38, RNAmmer38, EDGAR36, PEDANT 41, GeneMark 41, and NCBI Prokaryotic Genome Automated Annotation Pipeline (PGAAP) analysis 33, 36.
The following year also had several probiotic genomes including those of Lactobacillus rhamnosus 26, Lactobacillus vini 42, Lactobacillus curvatus 32, Lactobacillus fructivorans 43, and Lactobacillus helveticus 44 were sequenced using the Roche 454 GS FLX Titanium pyrosequencing technology, while the genome of Lactobacillus rossiae 34 was sequenced using the Illumina HiSeq 2000 platform. Even here, genome assembly was predominantly carried done using Newbler Assembler with the exception of the use of whole genome sequence assembler (wgs Assembler), genome sequence assembler (gsAssembler) and GS Reference Mapper for the assembly of Lactobacillus helveticus, Lactobacillus vini, and Lactobacillus rhamnosus genomes respectively 26, 42, 44. Genome annotation was done by similar software that was mentioned before with RAST and PGAAP being the predominant tools for annotation.
In 2013, probiotic genome sequencing witnessed a more heterogeneous usage of sequencing platforms with the Applied Biosystems ABI377 and 3700 automated sequencers 45, and the Ion Torrent Personal Genome Machine 46 entering the arena where Roche 454 GS FLX 47, Illumina Genome Analyzer Iix 48, and Illumina HiSeq 2000 49 existed. The species whose genomes were sequenced in this period include Lactobacillus pentosus 48, Lactobacillus helveticus 45, Lactobacillus shenzhenensis 49, Lactobacillus ginsenosidimutans 50, Lactobacillus florum 51, Lactobacillus pobuzihii 52, Lactobacillus jensenii, Lactobacillus gasseri 46, and Lactobacillus otakiensis 47. Additionally, with heterogeneous usage of sequencing technologies came the usage of multiple assembly and annotation software. While most sequences that came out of Roche 454 GS FLX platform were assembled by different versions of Newbler 47, sequence from Ion Torrent PGM were assembled using Ion Torrent Assembler 46 or CLC de Novo Genomics Workbench, while the output from Illumina platforms were assembled using SOAP deNovo 49 or Velvet 48 software. Annotation was predominantly done by RAST and PGAAP analysis, but ERGO, GTPS, RDP, Silva, and ERGO were the new additions to the group 45, 47.
The year 2014 witnessed an increased use of Illumina and Ion Torrent platforms for sequencing probiotic genomes. While genomes of Lactobacillus mucosae 53, Lactobacillus sakei 54, Bifidobacterium moukalabense 55, Lactobacillus sucicola 56, Lactobacillus farraginis 57, and Lactobacillus composti 57 were sequenced using Ion Torrent Personal Genome Machine, the genomes of Lactobacillus equi 58, Lactobacillus animalis 59, Lactobacillus oryzae, Lactobacillus fabifermentans 60, and Lactobacillus salivarius 61 were sequenced by Illumina platforms. Roche 454 GS FLX pyrosequencer was used to sequence Lactobacillus gasseri and Lactobacillus namurensis 62 genomes.
In case of genome assembly, there was a diverse use of assembly software that was perhaps used to match the requirements of a particular genome. While genomes sequenced using Roche 454 GS FLX continued to be assembled using Newbler assembler 62, genomes sequenced using Ion Torrent systems were assembled using Newbler 57, NGen (DNAStar) 53, or CLC Genomics Workbench 54, and reads from Illumina platforms used Abyss61, 63, Velvet 59, 63, Platanus 60, AMOS 59, Hawkeye 59 either in isolation or in concert. RAST server and PGAAP continued to be the predominant annotation platform, newer tools like GAMOLA59, MetaGene Annotator 60, MiGAP 60, SignalP 61, InterPro 61, TMHMM 61, and Artemis being used for annotation and curation.
In the next two years, a number of probiotic species were sequenced. The year 2015 not only witnessed the use of all types of sequencing technologies, but also witnessed the combinatorial use of many of them. The combinations were either a combination of Roche 454 pyrosequencers with Illumina platforms 64 or with Sanger sequencing methods 65. Single molecule real time (SMRT) Pacific Biosciences RSII sequencer was another technology that was used this year 66. The species that were sequenced during this year include Lactobacillus delbrueckii 67, Bifidobacterium catenulatum 68, Bifidobacterium pseudolongum66, Lactobacillus johnsonii 29, Lactobacillus rhamnosus 69, Lactobacillus reuteri 70, Bifidobacterium angulatum 71, Bifidobacterium adolescentis 71, Lactobacillus kunkeei 72, Lactobacillus mucosae 64, Bifidobacterium scardovii 65, Bifidobacterium aesculapii 73, Lactobacillus curieae 74, Lactobacillus acidophilus 75, Bifidobacterium actinocoloniiforme 76, Lactobacillus curvatus 77, Lactobacillus rhamnosus 69, Lactobacillus fermentum 78, 79, Bifidobacterium kashiwanohense 80, 81, Lactobacillus paracasei 82, Lactobacillus hokkaidonensis 83, and Lactobacillus farciminis 84. The assemblers used included Newbler 72, Velvet 29, gs Assembler 71, CLC Genomics Workbench 85, SOAP deNovo74, SPAdes 86, Ngen 67, and Phred-Phrap-Consed 68 as seen in the previous years and annotation was done mostly using RAST server and PGAAP pipeline 85, complemented with Glimmer, tRNAscan-SE, Prodigal, GenePRIMP 65, 72. One of the new assemblers used in this year was MIRA 64.
In 2016 also, several probiotic genomes have been sequenced mainly using Illumina platforms 87 with isolated use of Ion Torrent 88, Pacific BioSciences 35 and Roche 454 30 platforms as well. The probiotics that have been sequenced this year include Lactobacillus casei 30, 87, Lactobacillus sakei 89, Lactobacillus plantarum 88, 90, 91, Lactobacillus equigenerosi 92, Lactobacillus crispatus 93, Lactobacillus kunkeei 35, Bifido bacterium longum 94, Lactobacillus farciminis 95, Lactobacillus johnsonii 96, Lactobacillus brevis 97, and Lactobacillus collinoides 98. Genome assemblies were mostly done with the help of software like Newbler 92, Ngen 91, SOAP deNovo 96, SPAdes 88, Abyss 94, Ray Assembler 90, and CLC Genomics Workbench 87. Annotation was predominantly done using RAST server and PGAAP pipeline 91 with the additional use of Glimmer, tRNAscan-SE, and RNAmmer 91.
With the explosive amount of genomic data generated in the recent year, efforts towards their analyses have also been slowly progressing. The last two years have seen several comparative genomic analyses of the strains belonging to the aforementioned genera of probiotics 99 - 101.
Furthermore, in the recent years, a special interest is also observed in studies pertinent to carbohydrate utilization in these organisms 102. Also, there has been an impetus for the identification of novel genes helpful in diagnostics 103, and genomic characterization of important traits like motility77.
TABLE 1: SPECIES, TYPE OF GENOME SEQUENCE AND TECHNOLOGY USED
|Year||Species||Type of Genome sequence||Technology used|
|2004||Lactobacillus johnsonii NCC 533||Whole genome||Whole genome shotgun;
Annotation: tRNSscan-SE, COG, ORF,
|Lactobacillus paraplantarum C7||PLASMID|
|2005||Lactobacillus hilgardii 0006||Gene sequence|
|2009||Bifidobacterium animalis subsp. lactis AD011||Traditional Sanger paired end sequencing of
plasmid and fosmid libraries;
Assembly: PHRED, PHRAP, CONSED; Annotation: Glimmer, CRITICA; AUTOFACT; Artemis for annotation verification
|2011||Lactobacillus amylovorus GR1112
|Genome||454 GS FLX pyrosequencer;
Annotation: PGAP, EDGAR
|Lactobacillus amylovorus GR1118||Genome||454 GS FLX pyrosequencer;
|Lactobacillus ruminis SPM0211||Genome||454 GS FLX pyrosequencer; paired end; correction by Illumina IIx genome analyzer;
Assembler: GS deNovo Assembler 2.5 and CLC Genomics Workbench 4.5.1
|Lactobacillus iners AB-1|
|Lactobacillus coryniformis||Whole genome||shotgun 454 GS FLX; paired reads;
Assembler: Newbler 2.3;
Annotation; RAST, Glimmer 3.02, tRNAscan-SE, RNAmmer
|Lactobacillus cypricasei KCTC 13900||Genome||454 Titanium pyrosequencing (Roche);
Annotation: Glimmer3.02, RNAmmer1.2, RAST
|Lactobacillus coryniformis KCTC 3167||Genome||454 GS FLX pyrosequencer;whole genome shotgun; Assembler: Newbler2.3;
Annotation: RAST, Glimmer3.02, tRNAscan-SE 1.21, RNAmmer 1.2
|Lactobacillus animalis KCTC 3501||Genome||454 GS FLX pyrosequencer;whole genome shotgun; Assembler: Newbler2.3;
Annnotation: RAST, Glimmer3.02, tRNAscan-SE 1.21, RNAmmer 1.2
|Lactobacillus sanfranciscensis||Genome||Combined Sanger/454 pyrosequencing; Annnotation: PEDANT, GenMark2.8|
|Lactobacillus kefiranofaciens ZW3||Whole Genome||combo of 454 sequencing and GA IIx Solexa HTS; Assembler: Newbler;
Annotation: PHRED, PHRAP, CONSED, Glimmer, GenMark; Verification by Artemis
|2012||Bifidobacterium asteroids PRL 2011|
|Lactobacillus rhamnosus MTCC5462||Complete Genome||Shotgun; Roche GS 454;
Assembler: GS Reference Mapper v 2.3;
|Lactobacillus vini LMG 23202T, JP7.8.9||Genome||Roche 454 GS FLX Titanium;
|Lactobacillus curvatus CRL705||Draft||454 GS Titanium pyrosequencer;
Assembler: Newbler 2.5.3;
|Lactobacillus rossiae DSM 15814T||Genome||Shotgun Illumina sequencing HiSeq 2000; paired end;
|Lactobacillus fructivorans KCTC 3543||Genome||454 GS FLX Titanium pyrosequencer;
Assembler: Newbler 2.3;
Annnotation: RAST, Glimmer3.02, tRNAscan-SE 1.21, RNAmmer 1.2
|Lactobacillus helveticus R0052||Complete Genome||454 GS FLX Titanium;
Assembler: wgsAssembler v6.0;
|2013||Lactobacillus pentosus KCA1||Genome||Paired end Next Gen Illumina GAII sequencing; Assembly: VELVET assembler; Mauve and Artemis comparison tool|
|Lactobacillus helveticus CNRZ 32||Genome||Shotgun sequencing; Applied Biosystems ABI377 and 3700 automated sequencers; PE 377 automated DNA sequencers;
|Lactobacillus shenzhenensis strain
|Whole Genome||Illumina HiSeq 2000; paired end;
Assembler: SOAP deNovo 1.05;
Annotation: Glimmer 3.0, RAST
|Lactobacillus ginsenosidimutans sp|
|Lactobacillus florum||Draft||Paired end Illumina HiSeq 2000;
Assembler: Velvet 1.2.07;
|Lactobacillus pobuzihii E100301T||Draft||Illumina GAIIx;
|Lactobacillus jensenii MD IIE-70||Draft||Ion Torrent PGM;
Assembler: Ion Torrent Assembler and CLC Genomics Workbench deNovo assembler; Annotation: PGAP and RAST
|Lactobacillus gasseri Strain 2016||Draft||Ion Torrent PGM;
Assembler: Ion Torrent Assembler and CLC Genomics Workbench deNovo assembler
|Lactobacillus otakiensis JCM 15040 T||Whole Genome||454 GS FLX pyrosequencer; whole genome shotgun;
Assembler: Newbler 2.7;
Annotation: Glimmer3.02, GTPS, RDP, Silva, tRNAscan-SE
|2014||Lactobacillus gasseri K7||Improved Draft||454 GS FLX+;
Assembler: Newbler 2.6;
Annotation: PGAAP, IMG-ER; Artemis and IMG-ER for curation
|Lactobacillus mucosae CRL573||Draft||Whole genome shotgun Ion Torrent Personal Genome Machine (PGM);
Assmbler: NGen (DNAStar);
Annotation: PGAAP, tRNAscan-SE
|Lactobacillus sakei wikim 22||Draft||Ion Torrent and a 318 chip;
Assmbler: CLC Genomics Workbench v7.0.4; Validation of assembly by OSlay;
Annotation: GenemarkS, RNAmmer, tRNAscan, RAST
|Genome||GenProBio srl using Ion Torrent PGM|
|Lactobacillus salivarius||Draft||Illumina HiSeq2000;
Annotation: Glimmer3, GeneMark, Artemis, InterPro, SignaalP, TMHM
|Lactobacillus sucicola JCM 15457 T||Draft||Ion Torrent PGM system;
Assembler: Newbler v2.8;
Annotation: RAST, Glimmer3
|Lactobacillus fabifermentans T30PCM01||Genome||Illumina MiSeq;
Assembler: Abyss 1.3.6 and Velvet 1.2.10; Assemblies aligned using Mauve;
Annotation: RAST, GeneMark.hmm 2.8,
|Lactobacillus oryzae Strain SG293 T||Draft||Illumina MiSeq;
Assembler: Platanus v1.2.1;
Annotation: MiGAP, MetaGene Annotator 1.0, tRNAscan-SE 1.23, RNAmmer 1.2
|Lactobacillus animalis 381-IL-28||Draft||Illumina GAIIx and IonTorrent PGM;
Assembly: Velvet; manually validated with AMOS and Hawkeye;
Annotation: GAMOLA v2
|Lactobacillus namurensis Chizuka 01||Draft||Roche 454 GS FLX,
Assembler: Newbler 2.7;
|Lactobacillus equi||Genome||Illumina HiSeq2000;
|Lactobacillus gorilla sp. Nov.|
|L. farraginis JCM 14108 T||Draft||Ion Torrent PGM;
Assembler: Newbler v 2.8;
|L. composti JCM 14202 T||Draft||Ion Torrent PGM;
Assembler: Newbler v 2.8;
|2015||Lactobacillus delbrueckii subsp. bulgaricus CRL871||Draft
|Whole genome shotgun Ion Torrent (life technologies);
Assembler: Ngen (DNASTAR);
|Complete genome||Whole genome shotgun with sanger sequencing; Assembly: Phred-Phrap-Consed;
Annotation: Glimmer 3.0, tRNAscan-SE
|Bifidobacterium pseudolongum PV8-2||Genome||Single molecule real time (SMRT) PacBio RSII; Assembly: Heirarchical genome assembly process; Annotation: PGAP, RAST|
|Lactobacillus johnsonii strain 16||Draft||Illumina Genome analyzer IIx; paired ends; Assembler was Velvet0.7.54; Mapping MAQ0.7.1 and BWA 0.5.8c|
CNCM I -3698
|Draft||Illumina GAIIx; paired end;
Assembler: deNovo CLC Genomics Workbench 5.0;
Annotation: RAST and PGAP
|Bifidobacterium angulatum GT102||Draft||Whole genome shotgun Roche 454;
Assembler: gsAssembler v3.0
|Bifidobacterium adolescentis 150||Draft||Whole genome shotgun Roche 454; ;
Assembler: gsAssembler v3.0
|Lactobacillus kunkeei||Genome||454 GS FLX pyrosequencer Titanium;
Assembler: Newbler; Verified by BWA, Artemis, Artemis COMparison tool, Mauve;
Annotation: DIYA, Prodigal, tRNAscan, RNAmmer, genePRIMP
|Lactobacillus mucosae DPC 6426||Draft/Genome||454 GS FLX and Illumina MiSeq;
Assembly: MIRA; Artemis Comparison Tool; Annotation: RAST, Prodigal, Glimmer 3.02
|Complete Genome||Sanger and 454 GS FLX;
Assembly: Phred-Phrap-Consed, Newbler; Annotation: Glimmer 3.0, tRNAscan-SE
DSM 26737 T
Assembler: Newbler v 2.8;
|Lactobacillus kunkeei EFB6||HQ Draft||Genome Analyzer II (Illumina); paired end; Assembler: SPAdes 2.5;
Annotation: Glimmer3, YACOP, IMG-ER
CCTCC M 2011381 T
|Draft||Illumina Solexa HiSeq2000;
Assembler: SOAP deNovo;
Annotation: Glimmer 3, PGAP
|Lactobacillus acidophilus ATCC 4356||Draft||454 GS Titanium;
Assembly: Newbler v 2.6;
Annotation: RAST, PGAP
|Bifidobacterium actinocoloniiforme DSM 22766 T||Complete Genome||MiSeq and HiSeq 2000; paired end
Draft genome assembler: SPAdes v3.50 and A5 miseq; RAST
|Lactobacillus curvatus||Genome||HiSeq 2000:
Assembly: Velvet 1.2.07;
|Lactobacillus acidophilus FSI4||Complete Genome||Illumina GIIx; paired ends;
Assembler: Velvet; Error correction by Illumine HiSeq 2000
|Lactobacillus sp. strain TCF032-E4||Draft||Illumina HiSeq 2500; Contigs ordered by Mauve 2.3.1;
Assembler: Velvet 1.2.10;
|Lactobacillus rhamnosus CLS17||Draft||Roche 454 GS FLX Titanium;
Assembler: Newbler v 2.3;
|Lactobacillus rhamnosus||Draft||Roche 454 GS FLX Titanium;
Assembler: Newbler 2.6;
|Lactobacillus fermentum 3872||Genome||Ion Torrent PGM 314 v2 chip;
Assembler: Torrent Assembler and CLC Genomics Workbench combined using CISA contig integrator; Annotation: RAST, PGAP
|Complete Genome||WGS Sanger and 454 GS FLX pyrosequencing; Assembler: Newbler, Phred-Phrap-Consed; Annotation: Glimmer 3, tRNAscan-SE|
|Lactobacillus paracasei||Genome||Illumina Genome Analyzer II;
Assembler: Velvet deNovo;
Annotation: MiGAP, tRNAscan-SE
|Lactobacillus fermentum LfQi6||Draft||Illumina MiSeq;
Assembler: Velvet and SPAdes;
|Lactobacillus hokkaidonensis LOOC260(T)||Complete Genome||PacBio SMRT RSII sequencer; Also, independent Illumina MiSeq;
Assembly: deNovo by HGAP method, Platanus; Annotation: APBRO
|Genome||Illumina GAIIx; 454 GSFLX;
Assembly: CLC Genomics Workbench 5.0; Newbler 2.6;
Annotation: RAST, GO and Pfmagainst UFO web browser
|Bifidobacterium scardovii Strain
|Genome||Sanger and 454 GSFLX;
Annotation: Glimmer 3.0,
|Lactobacillus gorillae KZ01 T||Draft||Illumina MiSeq;
Assembler: CLC Genomics Workbench 8.0.1; Annotation: PGAP, ARDB
|Bifidobacterium kashiwanohense PV20-2||Complete Genome||SMRT PacBio RSII;
Assembly: Heirarchical genome assembly; Annotation: PGAP, RAST
|Lactobacillus curieae CCTCC M 2011381 T||Draft||Illumina SOlexa HiSeq 2000;
Assembler: SOAP deNovo;
Annotation: Glimmer 3.0, NCBI PGAP
|Lactobacillus plantarum P-8||Complete genome||454 GS FLX and Illumina Solexa GAIIx paired end combined;
|Lactobacillus panis DSM 6035 T||Draft||Illumina MiSeq;
|2016||Lactobacillus casei N87||Draft||Illumina HiSeq 1000;
Assembler: CLC Genomics Workbench v 8.0.3; Annotation: PGAP
|Lactobacillus sakei FBL1||Draft||Ion Torrent PGM;
Assembler: Ref based SPAdes v 3.1.0;
|Lactobacillus plantarum 2025||Draft||Ion Torrent PGM;
Assembler: SPAdes and GWB, consensus combined by CISA;
|Lactobacillus plantarum SF2A35B||Draft||WGS Illumina HiSeq 2000;
Assembly: deNovo by Ray Assembler;
Annotation: RAST server
|Lactobacillus plantarum CRL1506||Draft||WGS Illumina MiSeq;
Assembler: Ngen (DNAStar);
Annotation; RAST, PGAP, tRNAscan-SE; RNAmmer
NRIC 0697 T
Assembler: Newbler 2.8
|Lactobacillus crispatus JCM5810||Draft||Illumina MiSeq;
Assembler: CLC Genomics Workbench 8.5.1; scaffolds by Sanger seequencing
|Lactobacillus casei DPC6800||Draft||Roche 454 FLX;
Assembler: Ngen (DNAStar);
Annotation: Glimmer 3.0.2, RAS; verified by BLASTp and Artemis
|Lactobacillus kunkeei MP2||Genome||using one SMRT cell (P6-C4 Chemistry) on a PacBio RSII sequencer (Pacific Biosciences)|
|Bifidobacterium longum infantis TPY12-1||Illumina HiSeq2500; paired ends;
Annotation Abyss v.1.9.0
|Bifidobacterium longum suis BSM11-5||Illumina MiSeq; paired ends; annotation by RAST, Annotation Abyss v.1.9.0|
|Lactobacillus farciminis NBRC 111452||Draft||Ion Torrent PGM system;
Assembler: Newbler v2.8;
Annotation: RAST server using Glimmer3
|Lactobacillus johnsonii strain W1||Genome||Illumina MiSeq; paired ends;
Assembler: SOAP denovo 2.04.r240;
Annotation: PGAP analysis
|Lacobacillus brevis strain D6||Whole genome||Roche 454 GS FLX; Assembler: Newbler; Annotation: PGAAP analysis|
|Lactobacillus collinoides CUPV237||Draft||Illumina GAIIx;
Assembler: Genomics Workbench v 7.0; Annotation: PGAP
CONCLUSION: In conclusion, the application of genomic technologies in probiotic research has facilitated better understanding of probiotic bacteria and the genes and the molecular mechanisms that endow them with characteristic traits. The advances in sequencing technologies through the years, represented by the four generations of high throughput sequencing technologies, have eventually enabled easier and faster acquisition of genome data as seen by the reports of the genome sequences published over the years. A parallel advance has also been witnessed in the development of genome assembly and annotation software and tools to facilitate the analysis of the genome data. Furthermore, studies pertinent to the biomolecule utilization and comparative genomics studies of probiotic genomes have been gaining momentum in the recent years.
Future Work: As understanding complete genome maps of probiotic bacteria give us insights into the characteristic traits of particular species, it is important to analyze and understand the genomes of these probiotics. It is also crucial that we look deeper into the genome to see what they actually possess. Comparative Genomics studies have to be carried out as they could reveal genes that are critical in rendering the probiotics non-pathogenic, distinguishing them from the other bacteria. This will also help us connect the similar traits present in different probiotic species, helping us understand the evolutionary relationship among the bacterial communities that form the intestinal microbiota. It is therefore, the need of the hour to develop databases and tools that aid in the analysis of probiotic genomes through comparative genomics studies.
ACKNOWLEDGEMENT: The author would like to acknowledge the support of Department of Genetic Engineering, SRM University.
CONFLICT OF INTEREST: The author has no conflict of interest to declare.
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How to cite this article:
Joseph JP: Probiotic genomes: sequencing and annotation in the past decade. Int J Pharm Sci Res 2018; 9(4):1351-62.doi:10.13040/ IJPSR.0975-8232.9(4).1351-62.
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