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Comparative genomics of degradative Novosphingobium analysis pipeline

The pipeline is developed by by Juaning Wang (juanping_wang@163.com). For questions and comments, please contact Juanping or submit an issue on github.

The pipeline can be broadly separated into six main sections：

  1. The THN1 genome assembly, and annotation
  2. Identification of orthologous proteins
  3. Phylogenetic analysis
  4. Insertion sequence elements, genomic islands, and CRISPR detection
  5. Functional metabolic analysis and comparison
  6. Comparison analysis of the microcystin-degrading mlr gene cluster

1 The THN1 genome assembly, and annotation

1.1 Assembly

De novo assembly was performed using the SMRT Analysis pipeline v2.3.0 in conjunction with the HGAP assembler (Chien et al., 2016). Additional assemblies were performed using minimus2 (Treangen et al., 2011). The assembly was validated by aligning the raw reads onto the finished contigs using the BWA version 0.7.9a (Li and Durbin, 2009).

1.2 Annotation

Genes in the assembled genome were predicted using Prodigal(Hyatt et al., 2010). tRNA, rRNA, and ncRNA were identified by Infernal (Nawrocki et al., 2009) and RNAmmer (Lagesen et al., 2007). Protein-coding sequences were annotated based on BLASTP searches against the downloaded Rfam (Griffiths-Jones et al., 2005), NCBI NR, COG, KEGG (Kanehisa et al., 2008), and SwissProt (Watanabe and Harayama, 2001) databases with an E-value cut-off of 1e−20 and subsequent filtering for the best hits.

### Gene Prediction
~/software/prodigal.v2_60/prodigal  -i  THN1.fa -f gbk -o THN1 -a THN1_pro
### NCBI NR annotation
~/software/ncbi-blast-2.2.31+/binastp -num_threads 12 -query THN1_pro.fa -out THN1.csv -db nr -outfmt 7 -evalue 1e-10 -max_target_seqs 20

2 Identification of orthologous proteins

Groups of orthologous sequences were classified by clustering with OrthoMCL version 2.0.9 (Li et al., 2003) using a Markov cluster algorithm. Protein families were constructed using rule of 60%identity and 60% coverage in the alignments (Snipen and Ussery, 2010).

### InstallSchema
cp ~/doc/OrthoMCLEngine/Main/orthomcl.config.template ./ 
orthomclInstallSchema orthomcl.config.template orthomcl.config.log 
### AdjustFasta
orthomclAdjustFasta Aro ./proteins/Novosphingobium_aromaticivorans_DSM12444.faa 1
orthomclAdjustFasta Len ./proteins/Novosphingobium_lentum_NBRC107847.faa 1
                       ······
                       ......
                       ......
### FilterFasta
orthomclFilterFasta compliantFasta/ 10 20 
### blastp
makeblastdb -in goodProteins.fasta -dbtype prot -out good_proteins.fasta
blastp -db good_proteins.fasta -query goodProteins.fasta -outfmt 7 -out goodProteins_blastp.out
grep -v -P "^#" goodProteins_blastp.out > goodProteins_v1_blastp.out
### BlastParser,LoadBlast,Pairs,DumpPairsFiles
orthomclBlastParser goodProteins_v1_blastp.out ./compliantFasta >similarSequences.txt
orthomclLoadBlast orthomcl.config.template similarSequences.txt
orthomclPairs orthomcl.config.template pairs.log cleanup=no 
orthomclDumpPairsFiles orthomcl.config.template 
### mcl,MclToGroups
mcl mclInput --abc -I 1.5 -o mclOutput
orthomclMclToGroups No_ 1 <mclOutput >groups.txt

3 Phylogenetic analysis

3.1 Build 16S rRNA sequences phylogenetic trees

We performed a multiple sequence alignment of 16S rRNA sequences using Muscle(Edgar, 2004). Unaligned sequences were trimmed from the edges. Then,phylogenetic relationships based on the 16S rRNA sequences of the Novosphingobium strains were analyzed using MEGA v7.0.26 (Kumar et al., 2016) with the neighbor-joining method. The robustness of clustering was evaluated by 1000 bootstrap replicates.

3.2 Build core genes phylogenetic trees

The core genes were computed by OrthoMCLversion 2.0.9 (Li et al., 2003) and retrieved using inhouse scripts. We performed a multiple sequence alignment of the core genes using Clustal Omega(Sievers et al., 2011). The edge-trimmed core sequences were joined and the phylogenetic tree was constructed using MEGA v7.0.26.

Count_core.py
ExtractCoreSequence.pl
ClustalOmegaAlignment.sh
CoreEdgeTrim.sh
CoreJoin.pl

3.3 Calculate ANI values

Average nucleotide identity (ANI) values were calculated using the MUMmer algorithm of JSpecies v 1.2.1 (Kurtz et al., 2004; Richter andRossello-Mora, 2009; Chan et al., 2012) and visualized as a heatmap using Morpheus software (https://software.broadinstitute.org/morpheus/).

java -jar -Xms512m -Xmx512m ~/software/jspecies/jspecies1.2.1.jar

3.4 Build whole-genome-based phylogeny trees

We employed CVTree (version 3.9.6) (Qi et al., 2004) with a K value of 6 to compute the whole-genome composition vector of the 22 Novosphingobium strains. The robustness of this whole-genome-based phylogeny was validated by performing statistical resampling tests by 1000 bootstrap replicates using Seqkit (Shen et al., 2016). PHYLIP (Retief, 2000) was used to construct an eighbor-joining phylogenetic tree, which was visualized using MEGA v7.0.26(Kumar et al., 2016).

cvtree -i species.list -p data -o CVTree_k6.txt -k 6
neighbor

4 Insertion sequence elements, genomic islands, and CRISPR detection

Insertion sequences (ISs) were detected by BLAST comparisons (E-value ≤1e−5) against the ISFinder database (Siguier et al., 2006). IslandViewer 4 (Bertelli et al., 2017) was used to predict genomic islands (GIs), as described previously (Zhang et al., 2016). CRISPR arrays were detected using the CRISPRFinder (Grissa et al., 2007) online server to perform BLAST searches against dbCRISPR (CRISPR database).

5 Functional metabolic analysis and comparison

5.1 COG

We assigned the protein-coding genes to COG categories by searches against eggNOG version 4.5.1 (Huerta-Cepas et al., 2016) using the eggNOG-mapper tool(Huerta-Cepas et al., 2017).

python eggnog-mapper-1.0.3/emapper.py --cpu 30 -i total.fa  -d bact -o total

5.2 KEGG

Predicted proteins from the Novosphingobium genomes were also annotated using the KEGG KAAS server (Moriya et al., 2007). Metabolic pathways were deduced by manual inspection of KEGG Orthology (KO) that was predicted based on comparisons to the KEGG database (Kanehisa and Goto, 2000; Kanehisa et al., 2014).

6 Comparison analysis of the microcystin-degrading mlr gene cluster

We compared the mlr genes of each pair of strains by BLAST alignments. Codon usage of selected core genes and mlr genes was computed and analyzed by graphical codon usage analyser (http://gcua.schoedl.de) platform.

Datasets

Datasets/
├── Genomes
│   ├── Novosphingobium_aromaticivorans_DSM12444.fna
│   ├── Novosphingobium_barchaimii_LL02.fna
│   ├── Novosphingobium_lentum_NBRC_107847.fna
│   ├── Novosphingobium_lindaniclasticum_LE124.fna
│   ├── Novosphingobium_naphthalenivorans_NBRC_102051.fna
│   ├── Novosphingobium_panipatense_P5.fna
│   ├── Novosphingobium_pentaromativorans_US6-1.fna
│   ├── Novosphingobium_resinovorum_SA1.fna
│   ├── Novosphingobium_sp_63-713.fna
│   ├── Novosphingobium_sp_B-7.fna
│   ├── Novosphingobium_sp_Chol11.fna
│   ├── Novosphingobium_sp_Fuku2-ISO-50.fna
│   ├── Novosphingobium_sp_KN65.fna
│   ├── Novosphingobium_sp_MBES04.fna
│   ├── Novosphingobium_sp_P6W.fna
│   ├── Novosphingobium_sp_PC22D.fna
│   ├── Novosphingobium_sp_PP1Y.fna
│   ├── Novosphingobium_sp_SCN63-17.fna
│   ├── Novosphingobium_sp_SCN64-18.fna
│   ├── Novosphingobium_sp_ST904.fna
│   ├── Novosphingobium_sp_THN1.fna
│   └── Novosphingobium_subterraneum_NBRC16086.fna
└── Proteins
    ├── Novosphingobium_aromaticivorans_DSM12444.faa
    ├── Novosphingobium_barchaimii_LL02.faa
    ├── Novosphingobium_lentum_NBRC107847.faa
    ├── Novosphingobium_lindaniclasticum_LE124.faa
    ├── Novosphingobium_naphthalenivorans_NBRC_102051.faa
    ├── Novosphingobium_panipatense_P5.faa
    ├── Novosphingobium_pentaromativorans_US6-1.faa
    ├── Novosphingobium_resinovorum_SA1.faa
    ├── Novosphingobium_sp_63-713.faa
    ├── Novosphingobium_sp_B-7.faa
    ├── Novosphingobium_sp_Chol11.faa
    ├── Novosphingobium_sp_Fuku2-ISO-50.faa
    ├── Novosphingobium_sp_KN65.faa
    ├── Novosphingobium_sp_MBES04.faa
    ├── Novosphingobium_sp_P6W.faa
    ├── Novosphingobium_sp_PC22D.faa
    ├── Novosphingobium_sp_PP1Y.faa
    ├── Novosphingobium_sp_SCN63-17.faa
    ├── Novosphingobium_sp_SCN64-18.faa
    ├── Novosphingobium_sp_ST904.faa
    ├── Novosphingobium_sp_THN1.faa
    └── Novosphingobium_subterraneum_NBRC16086.faa

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Copyright (C) 2018 Juanping Wang.