To calculate the parameters -m (minimum) and -M (maximum), use the following formula:

Overlap = (2 × Reads) - Fragment

The minimum overlap (-m) is value between 10 or 20 bp, while the maximum overlap (-M) is determined by the calculated Overlap.

1. Read Length: Use the zcat command to inspect the raw file:

   zcat /home/marcos/PRJEB59406/fastq_files/ERR10856949_1.fastq.gz | head -n 2 | tail -n 1 | wc -c

2. Fragment Size: * Open the HTML report generated by fastp.

   • Look for the Insert Size Estimation graph.
   • Use the value indicated at the Peak.

graph TD
    %% Node Styles
    classDef shell fill:#e1f5fe,stroke:#01579b,stroke-width:2px,color:#000000;
    classDef python fill:#fff9c4,stroke:#fbc02d,stroke-width:2px,color:#000000;
    classDef ipynb fill:#e8f5e9,stroke:#2e7d32,stroke-width:2px,color:#000000;
    classDef start fill:#f3e5f5,stroke:#7b1fa2,stroke-width:2px,stroke-dasharray: 5 5,color:#000000;
    classDef refinement fill:#fff3e0,stroke:#e65100,stroke-width:2px,color:#000000;

    %% Start Node
    Input(Raw FASTQ Data):::start --> Clean1

    %% --- 1. CLEANING & ASSEMBLY ---
    subgraph Cleaning ["1. Cleaning & Assembly"]
        direction TB
        Clean1[fastp.sh]:::shell --> Clean2[align_reads_human_genome_Bowtie2.sh]:::shell
        Clean2 --> Clean3[Remove_human_genome_Bowtie2.sh]:::shell
        Clean3 --> Clean4[flash.sh]:::shell
        Clean4 --> Clean5[megahit.sh]:::shell
    end

    %% --- 2. TAXONOMY & BIN REFINEMENT ---
    Clean5 --> Tax1

    subgraph Taxonomy ["2. Taxonomy & Bin Refinement"]
        direction TB
        Tax1[indexing_contigs.sh]:::shell --> Tax2[align_contigs_read.sh]:::shell
        Tax2 --> Tax3[ordering_bam.sh]:::shell
        
        %% Multi-Binning
        Tax3 --> Bin1[metabat2.sh]:::shell
        Tax3 --> Bin2[semibin2.sh]:::shell
        Tax3 --> Bin3[comebin.sh]:::shell        
        
        %% Refinement
        Bin1 --> Refine[MAGScoT.R]:::refinement
        Bin2 --> Refine
        Bin3 --> Refine
        
        Refine --> Tax5[checkm2.sh]:::shell
        Tax5 --> Tax7[gtdb-tk.sh]:::shell
        Tax7 --> Tax8[mag_functional_screening.sh]:::shell
        Tax8 --> Tax9[create_master_table.py]:::python
    end
    
    %% --- 3. STATISTICAL ANALYSIS ---
    subgraph Stats ["3. Statistical Analysis"]
        direction TB
        Tax9 --> Stat1(plots_taxonomy.ipynb):::ipynb
    end

(Initial data setup and database downloads)

The pipeline quality-controls and decontaminates reads before assembly.:

1. fastp.sh (Quality Control)
2. align_reads_human_genome_Bowtie2.sh (Host Alignment)
3. Remove_human_genome_Bowtie2.sh (Decontamination)
4. flash.sh (Merge Paired-end Reads)
5. megahit.sh (Assembly)

1. Mapping: indexing_contigs.sh (Index Building), align_contigs_read.sh (Alignment), ordering_bam.sh (BAM Sorting)

2. Binning: metabat2.sh (MetaBAT2 Binning), semibin2.sh (SemiBin2 Binning), comebin.sh (Comebin Binning)

3. Refinement: MAGScoT.R (Bin Refinement)

4. Quality Assessment: checkm2.sh (CheckM2)

5. Taxonomic Classification: gtdb-tk.sh (GTDB-Tk)

6. Validation & Contamination Check (CAT/BAT):

• run_bat_pipeline.sh (Taxonomic Classification via Homology)
• merge_bat_results.py (Aggregate BAT Reports)

7. mag_functional_screening.sh (Targeted Functional Search)
8. create_master_table.py (Data Aggregation)

• plots_doc_simple

BUCHFINK, B.; XIE, C.; HUSON, D. H. Fast and sensitive protein alignment using DIAMOND. Nature Methods, v. 12, n. 1, p. 59–60, 17 nov. 2014.

CHAUMEIL, P.-A.; MUSSIG, A. J.; HUGENHOLTZ, P.; PARKS, D. H. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics, v. 38, n. 23, p. 5315–5316, 11 out. 2022.

CHEN, S. Ultrafast one‐pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta, v. 2, n. 2, 8 maio 2023.

DANECEK, P.; BONFIELD, J. K.; LIDDLE, J.; MARSHALL, J.; OHAN, V.; POLLARD, M. O.; WHITWHAM, A.; KEANE, T.; MCCARTHY, S. A.; DAVIES, R. M.; LI, H. Twelve years of SAMtools and BCFtools. GigaScience, v. 10, n. 2, 29 jan. 2021.

HARRIS, C. R.; MILLMAN, K. J.; VAN DER WALT, S. J.; GOMMERS, R.; VIRTANEN, P.; COURNAPEAU, D.; WIESER, E.; TAYLOR, J.; BERG, S.; SMITH, N. J.; KERN, R.; PICUS, M.; HOYER, S.; VAN KERKWIJK, M. H.; BRETT, M.; HALDANE, A.; DEL RÍO, J. F.; WIEBE, M.; PETERSON, P.; GÉRARD-MARCHANT, P. Array Programming with NumPy. Nature, v. 585, n. 7825, p. 357–362, 16 set. 2020. Disponível em: https://www.nature.com/articles/s41586-020-2649-2.

HYATT, D.; CHEN, G.-L.; LOCASCIO, P. F.; LAND, M. L.; LARIMER, F. W.; HAUSER, L. J. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics, v. 11, n. 1, 8 mar. 2010.

LANGMEAD, B.; WILKS, C.; ANTONESCU, V.; CHARLES, R. Scaling read aligners to hundreds of threads on general-purpose processors. Bioinformatics, v. 35, n. 3, p. 421–432, 18 jul. 2018.

LI, D.; LIU, C.-M.; LUO, R.; SADAKANE, K.; LAM, T.-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, v. 31, n. 10, p. 1674–1676, 20 jan. 2015. Disponível em: https://arxiv.org/pdf/1409.7208.pdf.

MAGOC, T.; SALZBERG, S. L. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, v. 27, n. 21, p. 2957–2963, 7 set. 2011.

PARKS, D. H.; IMELFORT, M.; SKENNERTON, C. T.; HUGENHOLTZ, P.; TYSON, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Research, v. 25, n. 7, p. 1043–1055, 14 maio 2015. Disponível em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484387/.

VIRTANEN, P.; GOMMERS, R.; OLIPHANT, T. E.; HABERLAND, M.; REDDY, T.; COURNAPEAU, D.; BUROVSKI, E.; PETERSON, P.; WECKESSER, W.; BRIGHT, J.; VAN DER WALT, S. J.; BRETT, M.; WILSON, J.; MILLMAN, K. J.; MAYOROV, N.; NELSON, A. R. J.; JONES, E.; KERN, R.; LARSON, E.; CAREY, C. J. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nature Methods, v. 17, n. 3, p. 261–272, 3 fev. 2020. Disponível em: https://www.nature.com/articles/s41592-019-0686-2.

VON MEIJENFELDT, F. A. B.; ARKHIPOVA, K.; CAMBUY, D. D.; COUTINHO, F. H.; DUTILH, B. E. Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT. Genome Biology, v. 20, n. 1, 22 out. 2019. Acesso em: 14 fev. 2022.