CSV file with summary of ambiguous/masked positions
The default output is a CSV file summarizing the masked positions per sample. You will find the information on how many positions were masked by the respective IUPAC base (N, W, S, M, K, R, Y, B, D, H, V) in masked_bases_summary.csv . Depth masking (N) is performed if fewer than ten bases are present at one position. The masked positions on the entire genome and only on the chromosome are shown for each sample.
For example:
In sample2, we have 3108 positions masked by N on all contigs (genome), which means these positions exhibit a sequencing depth below 10. Let's take a look only at the chromosome contig. We see a massive reduction in masked and especially depth-masked positions, so we used only the chromosomal contig for our analysis.
name,type,N(ATCG),W(AT),S(CG),M(AC),K(TG),R(AG),Y(TC),B(TCG),D(ATG),H(ATC),V(ACG)
sample1,genome,1,0,0,1,0,14,12,0,0,0,0,
sample1,chromosome,1,0,0,1,0,12,9,0,0,0,0,
sample2,genome,3108,100,88,109,110,396,405,0,0,0,0,
sample2,chromosome,19,0,2,0,4,200,181,0,0,0,0,
sample3,genome,9534,6,14,20,10,310,293,0,0,0,0,
sample3,chromosome,17,0,4,4,0,266,254,0,0,0,0,
Sequence Logo
A graphical presentation of all ambiguous positions and their surrounding bases per analyzed sample. The overall height of each stack indicates the sequence conservation at a position (measured in bits)
, whereas the height of symbols within the stack reflects the relative frequency of the corresponding
base at that position.
In default mode, the sequence logo shows five bases up and downstream of the ambiguous position masked by IUPAC-Code. The length of the logo can be modified using the --motif flag.
For example:
For sample BK16641_k14 we have 233 masked positions by R and 225 masked positions by Y. For all reagions masked by R and Y we see rough conserved motives RACG and CGTY.
Frequency Plot
A violin chart is created when the --frequency flag is used. The plot is build from all analyzed samples of the run. For each ambiguous position found by aligning reads to their reference, the percentage occurrence of the bases within the reads is calculated. The orientation of the strand (forward or reverse) is determined and displayed as data points in two plots per position.
For example:
This plot comprises 6,556 ambiguous positions from 33 samples. Each dot represents a base occurrence within the respective base combination at the ambiguous position. The main errors exist when the Basecaller need to decide between Purin bases (A and G) masked by IUPAC Code R and Pyrimidin bases (T and C) masked by IUPAC code Y. 3,311 positions were masked by R. The plot differntiate between forward and reverse strand. On the reverse strand Guanine is more frequently called (100-95%) as Adenine (0-5%). This leads to the conclusion that Guanine is the correct base in this position. It's not that clear on the forward strand. The basecaller mainly calles Adenine in this position and less frequent Guanine. This base ratio in the reads can results in erroneous assemblies. Since it is a strand-specific error, it points to base-modification.
Citation
Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors
Mara Lohde, Gabriel E. Wagner, Johanna Dabernig-Heinz, Adrian Viehweger, Sascha D. Braun, Stefan Monecke,
Celia Diezel, Claudia Stein, Mike Marquet, Ralf Ehricht, Mathias W. Pletz, Christian Brandt
doi.org/10.1101/2023.09.15.556300
If you are interested in the work of our group: click here
FAQ
Maybe we can help you.
This is a RAM related error.
This means your computer did run out of Memory. You could try in reducing the overall parallelistation of MPOA by adding the following flags for instance --cores 8 --max_cores 8 This will execute only one process after another and assigning only 8 threads to each. This should execute less process simultaneously. MPOA was tested on a 16 GB RAM laptop with 8 threads and was working fine.
MPOA Releases If you want to run a specific release version of MPOA, use the -r flag and the version you want to execute. For example: nextflow run replikation/MPOA --fasta '*.fasta' --fastq '*.fastq.gz' -profile local,docker -r '1.4.1'
Head over to MPOA`s issue sections Use search first, maybe someone had the same issue already. If not create a new issue.
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