Based on a detailed analysis of the BLASTn results (combined with the depth/coverage data you provided in your very first message), here is the classification of your contigs.
Crucial Insight: You cannot rely solely on sequencing depth (coverage) to distinguish plasmids from chromosomes. High-depth contigs are often multi-copy chromosomal repetitive elements (like rRNA operons, insertion sequences, or transposons), while some true plasmids may show low depth due to assembly fragmentation. The Top BLAST Hits (Rank 1-5) are the ultimate judge.
Here is the definitive breakdown:
🟢 1. Confirmed Plasmids
These contigs have top BLAST hits (100% Query Cover, ~100% Identity) that explicitly match known plasmids.
- contig47 (Depth: 2.20x) -> Top hit: plasmid pDETABR21-1
- contig49 (Depth: 5.25x,
circular=true) -> Top hit: plasmid pRAB57-5 - contig50 (Depth: 2.28x) -> Top hit: plasmid unnamed2
- contig51 (Depth: 6.09x,
circular=true) -> Top hit: plasmid unnamed3 - contig61 (Depth: 3.94x) -> Top hit: plasmid pDETABR21-1
🔴 2. Confirmed Chromosomal Contigs (or Chromosomal MGEs)
These contigs have top BLAST hits that explicitly match chromosomes / complete genomes.
- contig53 (Depth: 10.60x) -> Top hit: chromosome
- contig58 (Depth: 3.92x) -> Top hit: chromosome
- contig63 (Depth: 1.94x) -> Top hit: chromosome
- contig64 (Depth: 2.52x) -> Top hit: chromosome
- contig65 (Depth: 7.40x) -> Top hit: chromosome
- contig66 (Depth: 18.10x) -> Top hit: chromosome
- contig67 (Depth: 1.71x) -> Top hit: chromosome
⚠️ 3. The “Depth Traps” (Very Important)
Some of your contigs show a mismatch between their sequencing depth and their BLAST identity. This is a classic bioinformatics scenario:
- The “False Plasmid” Trap (High Depth, but Chromosomal):
- contig48 (Depth: 7.50x) and contig53 (Depth: 10.60x).
- Analysis: Despite having very high coverage (which usually suggests plasmids), their BLAST results show 100% identity to the chromosome. This means they are not plasmids. They are multi-copy chromosomal regions (e.g., Insertion Sequences (IS), transposons, or rRNA operons) that the assembler separated into distinct contigs because they are repetitive. Do not flag these as plasmids.
- The “False Chromosome” Trap (Low Depth, but Plasmid):
- contig54 (Depth: 0.51x).
- Analysis: The depth is unusually low, but the top BLAST hits are 100% identical to known plasmids. This is a true plasmid. The low depth likely occurred because the assembler struggled with repetitive regions on the plasmid, breaking it into smaller pieces, or it is a very low-copy / mobilized plasmid. Treat this as a plasmid.
🧬 4. The Ambiguous / Mobile Genetic Element (MGE)
- contig59 (Depth: 6.31x)
- Analysis: The BLAST results are a mix of
chromosome(Rank 1, 2, 7, 9) and massiveplasmids(Rank 3, 4, 5, 6, 8 – many of which are >200kb NDM-carrier plasmids). - Conclusion: This 1.2kb sequence is likely a Mobile Genetic Element (MGE), such as a transposon (e.g., Tn125 carrying an AMR gene) or an Integrative and Conjugative Element (ICE). These elements can exist integrated into the chromosome and be found on large conjugative plasmids. Given the 6.3x depth, it is likely a multi-copy chromosomal transposon. NCBI’s PGAP will likely annotate it as a chromosomal feature or an unclassified MGE.
- Analysis: The BLAST results are a mix of
💡 Summary for your NCBI Submission:
When you submit your filtered FASTA file to the NCBI PGAP:
- Keep the
circular=trueflag only for contig49 and contig51 (as you manually verified and the BLAST confirms). - Do not add
circular=trueto the other plasmids (47, 50, 61) unless you are absolutely certain they are circular, as linear plasmids or fragmented plasmid contigs are perfectly acceptable for WGS submissions. PGAP will annotate them asplasmidbased on the gene content (e.g., repA, mob genes). - Submit all the chromosomal contigs (53, 58, 63-67, and the multi-copy traps 48, 53, 59) as part of the main genome assembly. PGAP is smart enough to recognize repetitive chromosomal elements.