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Configuration
In the following the parameters in the config.yml are discussed. In principle, the config.yml consists of different blocks. ScanNeo2 always utilizes the config.yml that is located in config/config.yml. However, the option --configfile allows specifying custom config files. It is to be noted that merely overwrites the config/config.yml and should therefore include all parameters to prevent using settings from multiple config files.
On the top level, the parameters are applied system-wide when applicable. This includes the reference genome within the reference attribute. The release option corresponds to the ENSEMBL release version and nonchr indicates to whether (=true) or not (=false) DNA sequence that is not assigned to chromosomes should be included in the analysis. Other options include the number of cores (threads), the mapping quality (mapq), and the average Phred scores (basequal).
threads: 30
mapq: 30
basequal: 20
The data block contains the sequencing reads specified as the indented blocks name, dnaseq, and rnaseq.
data:
name: <name/of/sample>
dnaseq:
<group1>: <path/to/dnaseq/reads1> [path/to/dnaseq/reads2]
<group2>: <path/to/dnaseq/reads1> [path/to/dnaseq/reads2]
rnaseq:
<group1>: <path/to/rnaseq/reads1> [path/to/rnaseq/reads2]
normal: <group2>
custom:
variants:
hlatyping:
MHC-I:
MHC-II:
readgroups:
The name key-value pair contains the name of the sample. This is also the name of the folder in which the analysis results are stored (e.g., results/<name/of/sample>). The blocks dnaseq and rnaseq specify the paths to the sequencing reads. In the <group1>:<path/to/dnaseq/data> key-value pair, the path to the DNA-seq data is defined. This can be either in .bam or .fastq. In the case of paired-end reads, forward and reverse read need to be separated by space. Similarly, rnaseq: <path/to/rnaseq/data> defines the RNA-seq data. Scanneo2 allows to specify multiple samples, using the same identation within the dnaseq or rnaseq blocks (e.g., ). These can correspond to readgroups or conditions. However, these need to be unique. In addition, normal allows to specify normal samples but is not used currently. Multiple normal samples can be separated by spaces. Scanneo2 operates on both RNA-seq and DNA-seq, but in principle also works with either DNA-seq or RNA-seq data. However, providing only DNA-seq data is restricted to detecting indels and SNVs.
In addition, the custom block allows the (optional) specification of user-defined data.
In variants predefined variants in VCF format can be provided. When available ScanNeo2 utilizes specific INFO keys, which are used in the results. These include AO, DP, AF which correspond to the observed alleles (supporting reads), the depth of the variant, and the variant allele frequency, respectively.
In the hlatyping property, user-defined class I (MHC-I) and class II (MHC-II) alleles can be provided in tab-delimited format. See the hla section in the output wiki page for more information.
It is to be noted that the custom block allows to specify additional information for the analysis. In other words, ScanNeo2 utilizes these files to augment the actual analysis, unless other options are deactivated (e.g., hlatyping, indel,...)
preproc:
activate: true
minlen: 10
slidingwindow:
activate: true
wsize: 3
ScanNeo2 provides an optional pre-processing procedure that is only applied to raw sequencing data. Here, activate: true enables the pre-processing, that can be combined with a window trimming from the 3'-end with a defined window size (wsize). Other parameters include the minimum length of the sequencing reads (minlen). Note: the globally defined base quality is also applied here.
align:
chimSegmentMin: 20
chimScoreMin: 10
chimJunctionOverhangMin: 10
chimScoreDropMax: 30
chimScoreSeparation: 10
In principle, the alignment procedure is done differently for DNA- and RNA-seq data. In the case of DNA-seq, the reads are directly aligned using BWA. For RNA-seq data, the sequencing reads are first aligned using STAR followed by realignment with BWA. The reason for that is the variant calling on the transcriptome (e.g., gene fusion, alternative splicing, exitron) which requires splice-aware alignments. Consequently, the parameters in this section control the chimeric alignments and are identical to STAR v2.7.10b. Please refer to the STAR manual for details.
| Option | Description |
|---|---|
chimSegmentMin |
minimum length of chimeric segment length, if ==0, no chimeric output |
chimScoreMin |
minimum total (summed) score of the chimeric segments |
chimJunctionOverhangMin |
minimum overhang for a chimeric junction |
chimScoreDropMax |
max drop (difference) of the chimeric score (the sum of scores of all chimeric segments) from the read length |
chimScoreSeparation |
minimum difference (separation) between the best chimeric score and the next one |
Each module in the variant calling can be switched on/off using the activate (true or false) property.
altsplicing:
activate: true
confidence: 3
iterations: 5
edgelimit: 250
In the detection of alternative splicing events, the parameter confidence determines how strongly input alignments are filtered before new nodes and edges are added to the splicing graphs. There are four confidence levels, with confidence increasing from 0 to 3. iterations add new intron edges into the splicing a certain number of times. Increasing the value increases the sensitivity, but also the runtime. Shouldn't be set lower than 5. edgelimit sets an upper boundary for the maximum number of edges (to reduce its complexity) and limit the runtime.
| Parameter | Value | Description |
|---|---|---|
confidence |
0-3 | Confidence Interval for the SplAdder with 0 the lowest and 3 the highest confidence |
iterations |
5- | Number of iterations to add new intron edges into the splicing graph |
edgelimit |
250- | Limit the number of edges in the splicing graph |
Please refer to the SplAdder documentation for more details.
exitronsplicing:
activate: true
ao: 3
pso: 0.05
strand: 1 # 0=unstranded, 1=forward, 2=reverse
In the exitron splicing, the reported exitrons are controlled by ao (allele observed) and pso (percent spliced in). The former describes the minimum number of reads that support the exitron, and the latter the minimum cutoff for the exon-exclusion rate. In other words, the ratio of the relative abundance of all isoforms missing a certain exon over the relative abundance of all isoforms of the gene missing the exon.
| Parameter | Value | Description |
|---|---|---|
| ao | 0- | Minimum number of supporting reads for an exitron event |
| pso | 0.0-1.0 | Minimum cutoff for the percent spliced out index |
genefusion:
activate: true
maxevalue: 0.3
suppreads: 2
maxsuppreads: 1000
maxidentity: 0.3
hpolymerlen: 6
readthroughdist: 10000
minanchorlen: 20
splicedevents: 4
maxkmer: 0.6
fraglen: 200
maxmismatch: 0.01
In the detection of gene fusion events, ScanNeo2 utilizes Arriba. Please refer to the tools website for more information. ScanNeo2 allows to specify the following parameters. maxevalue is a cutoff for the the number of supporting reads that are expected to have occurred by chance (e-value). Fusion events that exceed this cutoff are discarded. It is to be noted that a high e-value both reports more false positives and increases the runtime dramatically. suppreads is a cutoff for the minimal number of supporting reads. This means that fusion events that fall short of this value are discarded. Similarly, maxsuppreads defines the maximal number of supporting reads for fusion events which are discarded when exceeding this value. Arriba issues a warning when the threshold has been hit (see logs).
indel:
activate: true
mode: BOTH
strategy: OPTIMAL_F_SCORE # OPTIMAL_F_SCORE, FALSE_DISCOVERY_RATE, CONSTANT
fscorebeta: 1.0
fdr: 0.05
sliplen: 8
sliprate: 0.1
hlatyping:
class: I # I, II or BOTH
# the mode (origin) for the genotyping of each class (comma-separated list)
MHC-I_mode: RNA # DNA, RNA, custom (if empty alleles have to be specified in custom)
MHC-II_mode: RNA # DNA, RNA, custom (if empty alleles have to be specified in custom)
# specific path for class II hlatyping (only required when class: II, or BOTH)
freqdata: ./hlahd_files/freq_data/
split: ./hlahd_files/HLA_gene.split.txt
dict: ./hlahd_files/dictionary/
prioritization:
class: I # I, II or BOTH
lengths:
MHC-I: 8,9,10,11
MHC-II: 13,14,15