Dear Biostars,

I am posting this question in this forum because I found this wonderful site is populated mostly by very kind and helpful people

I am a beginner on the field of bioinformatics and genome assembly.

I am working on assembling several bacterial genomes. The sequences were obtained from pacbio. I found that there may be some contamination in the sequences of the bacteria I am trying to assemble. I used Minimap2 to align potential contaminants to the bam files (converted to fastq) . I used as reference for minimap2 the genomes of potential contaminants.

For example, i am trying to assemble a genome from the genus Ignatzschineria (Ignatz). The potential contaminants are: Moellerella (Moell) , Kurthia, Providencia (Prov). I also mapped the query to two genomes from the genus Ignatzschineria.

An example of the command line I used to align the fastq to a reference genome is:

minimap2 -ax map-pb KurthiaSp11kri321_CG_RefSeq_NZ_CP013217_1.fasta m54092_180525_143105.subreadsFQ.fastq > KurthiaRefVsIgnatz_alignmentpacbio.sam

The stderr and stdout from minimap2 seem to me unusable (am I correct?)

After the alignment, I used samtools to obtain the flagstas for each minimpa2-outputed .sam file: For example:

samtools flagstat KurthiaRefVsIgnatz_alignmentpacbio.sam

I need your help to interpret the results I obtained from flagstats for the 6 reference genomes:

samtools flagstat MoellRefVsIgnatz_alignmentpacbio.sam              
740728 + 0 in total (QC-passed reads + QC-failed reads)             
5523 + 0 secondary              
129702 + 0 supplementary                
0 + 0 duplicates                
599270 + 0 mapped (80.90% : N/A)                
0 + 0 paired in sequencing              
0 + 0 read1             
0 + 0 read2             
0 + 0 properly paired (N/A : N/A)               
0 + 0 with itself and mate mapped               
0 + 0 singletons (N/A : N/A)                
0 + 0 with mate mapped to a different chr               
0 + 0 with mate mapped to a different chr (mapQ>=5)

samtools flagstat KurthiaRefVsIgnatz_alignmentpacbio.sam                
621596 + 0 in total (QC-passed reads + QC-failed reads)             
15296 + 0 secondary             
797 + 0 supplementary               
0 + 0 duplicates                
20226 + 0 mapped (3.25% : N/A)              
0 + 0 paired in sequencing              
0 + 0 read1             
0 + 0 read2             
0 + 0 properly paired (N/A : N/A)               
0 + 0 with itself and mate mapped               
0 + 0 singletons (N/A : N/A)                
0 + 0 with mate mapped to a different chr               
0 + 0 with mate mapped to a different chr (mapQ>=5)

samtools flagstat IgnatzUreRefVsIgnatz_alignmentpacbio.sam          
613190 + 0 in total (QC-passed reads + QC-failed reads)         
4455 + 0 secondary          
3232 + 0 supplementary          
0 + 0 duplicates            
15101 + 0 mapped (2.46% : N/A)          
0 + 0 paired in sequencing          
0 + 0 read1         
0 + 0 read2         
0 + 0 properly paired (N/A : N/A)           
0 + 0 with itself and mate mapped           
0 + 0 singletons (N/A : N/A)            
0 + 0 with mate mapped to a different chr           
0 + 0 with mate mapped to a different chr (mapQ>=5)         

samtools flagstat IgnatzLARVAERefVsIgnatz_alignmentpacbio.sam           
609223 + 0 in total (QC-passed reads + QC-failed reads)         
3430 + 0 secondary          
290 + 0 supplementary           
0 + 0 duplicates            
8303 + 0 mapped (1.36% : N/A)           
0 + 0 paired in sequencing          
0 + 0 read1         
0 + 0 read2         
0 + 0 properly paired (N/A : N/A)           
0 + 0 with itself and mate mapped           
0 + 0 singletons (N/A : N/A)            
0 + 0 with mate mapped to a different chr           
0 + 0 with mate mapped to a different chr (mapQ>=5)         

samtools flagstat ProvALCALRefVsIgnatz_alignmentpacbio.sam
664124 + 0 in total (QC-passed reads + QC-failed reads)
49931 + 0 secondary
8690 + 0 supplementary
0 + 0 duplicates
129215 + 0 mapped (19.46% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

samtools flagstat ProvRETTRefVsIgnatz_alignmentpacbio.sam
659123 + 0 in total (QC-passed reads + QC-failed reads)
12368 + 0 secondary
41252 + 0 supplementary
0 + 0 duplicates
133975 + 0 mapped (20.33% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

I believe the results above indicated that Ignatz may not be Ignatz, but Moellerella, numeral "1." (80% of the queried sequences map to this genus).

Also, the 2 Ignatzschineria used as reference, IgnatzUre (numeral "3.") and IgnatzLarvae (numeral ".4") produce a low % of queried sequences mapping to them.

The 2 Providencia show about 20% of the queried sequences mapping to them. This may reflect their phylogenetic relationship.

Is my interpretation correct?

Regards,

Cecilio