Annotate-a-genome

Project Goals

A Borrelia Phylogeny

• Annotate and add newly sequenced Borrelia genomes to BorreliaBase
• Build an informatics pipeline for gene prediction, ortholog calls, databasing, and synteny analysis

Claim your assigned genome

Genome_id	Strain	Species	Group	Genome Sequences	Notes
100	B31	B. burgdorferi (reference genome)	Lyme Disease	main chromosome cp26 plasmid lp54 plasmid	Reference. Already downloaded as "ref.pep"
114	CA382	B. burgdorferi (California)	Lyme Disease	main chromosome	Accession: CP005925; Assigned to: HA
115	CA8	B. burgdorferi (California)	Lyme Disease	7 unassembled contigs	Accession: ADMY01000001; Assigned to: AA Accession: ADMY01000002; Assigned to: TAA Accession: ADMY01000003; Assigned to: KD Accession: ADMY01000004; Assigned to: JG Accession: ADMY01000005; Assigned to: KPG Accession: ADMY01000006; Assigned to: GG Accession: ADMY01000007; Assigned to: TDH
304	BgVir	B. garinii (Russia)	Lyme Disease	main chromosome cp26 lp54	Accession: CP003151; Assigned to: LH Accession: CP003201; Assigned to: SK Accession: CP003202; Assigned to: BK
305	NMJW1	B. garinii (China)	Lyme Disease	main chromosome	Accession: CP003866; Assigned to: AL
402	HLJ01	B. afzelii (China)	Lyme Disease	main chromosome	Accession: CP003882; Assigned to: RL
1003	Ly	B. duttonii (Tanzania)	Relapsing Fever	main chromosome lp23 (homolog of cp26 in LD genomes) Many other plasmids (not to include)	Accession: CP000976; Assigned to: HL Accession: CP000980; Assigned to: NM
1001	A1	B. recurrentis (Ethiopia)	Relapsing Fever	main chromosome lp124 (homologous to lp54 in LD genomes) lp23	Accession: CP000993; Assigned to: JP Accession: CP000994; Assigned to: DP Accession: CP000995; Assigned to: GAR
1100	DAH	B. hermsii (Washington State)	Relapsing Fever	main chromosome	Accession: CP000048; Assigned to: KR
1101	MTW	B. hermsii (?)	Relapsing Fever	main chromosome	Accession: CP005680; Assigned to: ?
1102	YBT	B. hermsii (?)	Relapsing Fever	main chromosome	Accession: CP005706; Assigned to: ?
??	SLO	B. parkeri (?)	Relapsing Fever	main chromosome	Accession: CP005706; Assigned to: ?
1103	YOR	B. hermsii (?)	Relapsing Fever	main chromosome	Accession: CP004146; Assigned to: ?
1200	91E135	B. turicatae (Texas)	Relapsing Fever	main chromosome	Accession: CP000049; Assigned to: MDR
1002	Achema	B. crocidurae (Mauritania)	Relapsing Fever	main chromosome Many unassembled plasmids (not to include)	Accession: CP003426; Assigned to: VS
1400	HR1	B. parkeri (??)	Relapsing Fever	main chromosome	Accession: CP0007022; Assigned to: AV
1300	LB-2001	B. miyamotoi (Northeast US)	Relapsing Fever	main chromosome	Accession: CP006647; Assigned to: LLW
107	94a	B. burgdorferi (Northeast US)	Lyme Disease	main chromosome	Accession: ABGK02000008; Assigned to: QZ

Protocol

Dependencies

• BASH (default shell of Linux OS and Apple OS X)
• Perl and BioPerl
• DNATweezer
• NCBI Standalone BLAST+

Part 1. Fetch genome sequences and extract protein sequences

• Note: These scripts are in "../../bio425/annotate-a-genome-pipeline". You may either make a copy to your home directory (recommended) or run directly from that directory by including the path
• Commands:

./fetch-genome.pl <your_assigned_accession> # Expected output: "accession.gb"
./gb2pep.pl <accession.gb> # Expected output: "accession.pep"

Part 2. Predict orthologs with reciprocal BLAST

• Note 1: Replace the "accession" in the following commands with your assigned accession number.
• Note 2: You may have to lower the identity (-d, default 90) and length coverage (-l, default 90) for replapsing fever genomes
• Commands:

cp ../../bio425/annotate-genome-pipeline/b31.pep . # get reference genome
makeblastdb -in b31.pep -dbtype 'prot' -parse_seqids -out ref # Prepare the ref genome DB
makeblastdb -in accession.pep -dbtype 'prot' -parse_seqids -out mygenome # Prepare the new genome DB
blastp -query accession.pep -db ref -outfmt '6 qseqid sseqid pident length qlen evalue' -evalue 1e-3 -out accession.fwd # Forward BLAST # customized outfmt 6
blastp -query b31.pep -db mygenome -outfmt '6 qseqid sseqid pident length qlen evalue' -evalue 1e-3 -out accession.rev # Reverse BLAST
./find-reciprocal.pl <accession.fwd> <accession.rev> > accession.orthlogs 2> accession.not-orthologs # Identify orthologs. 
# check results. If orthologs less than 90% of total ORFs in genbank file, run the previous command with more relaxed stringency (use "-d 80", 80% identify cutoff)
wc accession.orthlogs
wc accession.not-orthologs

Part 3. Generate database tables & deposit your results

• Note: Use your assigned genome_id in the table above as the argument for the "-g" option

./gb2table.pl -g <genome_id> -c <accession.gb> # Expected output: "accession.contig.txt"
./gb2table.pl -g <genome_id> -f <accession.gb> # Expected output: "accession.orf.txt"
# Check your outputs
wc <accession.contig.txt>
head <accession.contig.txt>
tail <accession.contig.txt>
wc <accession.orf.txt>
head <accession.orf.txt>
tail <accession.orf.txt>
cp accession.contig.txt ../../bio425/annotate-a-genome-results/your_initial.accession.contig.txt
cp accession.orf.txt ../../bio425/annotate-a-genome-results/your_initial.accession.orf.txt
cp accession.orthologs ../../bio425/annotate-a-genome-results/your_initial.accession.orth
cp accession.not-orthologs ../../bio425/annotate-a-genome-results/your_initial.accession.not-orth