Modules documentation¶
AllMine is developed in a modular fashion. Current AllMine modules are documented here. Some of them include tweakable parameters ! Only key steps modules are documented here.
fastp; reads presprocessing¶
AllMine uses fastp v0.20.0 to perform presprocessing on submited reads. Two modules, fastp_pe and fastp_se have been implemented to handle paired end and single end reads respectively.
Parameters :
--correction: Enable paired end overlaping regions correction.--cut_mean_quality: Set to 20. Threshold for trimming in both 5’ and 3’. You can increase this number for a more stringeant quality trimming.--cut_window_size: Size of the sliding window for sliding window trimming. Increasing this number will relax the trimming. Set to 1 for per base trimming (not recommended).--complexity_threshold: Set to 30. Complexity is defined as P(base[i] != base[i+1]). Reads bellow the threshold are discarded. Usefull to filter polyA tails in RNA seq data !-w: Threads used.--max_len1 350: Maximum lenght of submited reads. Longer reads will be trimmed from 3’ end to the maximum size.
Please refer to the fastp manual for more informations.
FastQC; quality control¶
AllMine uses FastQC v0.11.8 to perform quality control on submited reads. Two modules, fastqc_pe and fastqc_se have been implemented to handle paired end and single end reads respectively. Numerous indices and statistics are computed by FastQC. You may inspect them to ensure your data quality.
BWA index; genome indexing¶
If you have submited DNAseq data, AllMine will index your reference genome using
the bwa index command from BWA v0.7.17.
Parameters :
-a is: Algorithm used to index the genome. IS does not work for large genome (size > 3Gbp). Switch to-a bwtswif needed.
STAR index; genome indexing¶
If you have submited RNAseq data, AllMine will index your reference genome using
the STAR --runMode genomeGenerate command from
STAR v2.7.0f.
To perfom a two pass mapping strategy
BWA mem; DNAseq read mapping¶
If you have submited DNAseq data, AllMine will map your reads using
the bwa mem command from BWA v0.7.17.
Two modules, bwa_pe and bwa_se have been implemented to handle
paired end and single end reads respectively. This mapping algorithm handle
reads from 70bp to 1Mbp.
Parameters :
-w: Set to 100 bp. Band width. Essentially, gaps longer than INT will not be found.-d: Set to 100. Z-dropoff. Avoids unnecessary extension, but also reduces poor alignments inside a long good alignment.-r: Set to 1.5. Larger value yields fewer seeds, which leads to faster alignment speed but lower accuracy.-B: Set to 4. Mismatch penalty.-O: Set to 6. Gap opening penalty.-E: Set to 1. Gap extention penalty.-L: Set to 5. When performing SW extension, BWA-MEM keeps track of the best score reaching the end of query.
Note : In most of the cases BWA parameters are well suited for balance between accuracy and computational cost.
STAR; RNAseq reads mapping¶
If you have submited RNAseq data, AllMine will map your reads using STAR
command from STAR v2.7.0f. AllMine apply
a two pass mapping strategy. Two both handle paired and single end data four
modules where developped star_pe_FP, star_pe_SP, star_pe_FP and
star_pe_SP.
Parameters :
--scoreGap: Set to 0. Gap penalty.--scoreGapNoncan: Set to -8. Non-canonical junction penalty.--scoreGapGCAG: Set to -4. GC/AG and CT/GC junction penalty.--scoreGapATAC: Set to -8. AT/AC and GT/AT junction penalty.--scoreGenomicLengthLog2scale: Set to -0.25. Extra score logarithmically scaled with genomic length of the alignment : scoreGenomicLengthLog2scale*log2(genomicLength).--scoreDelOpen: Set to -2. Deletion open penalty.--scoreDelBase: Set to -2. Deletion extension penalty per base (in addition to scoreDelOpen).--scoreInsOpen: Set to -2. Insertion open penalty.--scoreInsBase: Set to -2. Insertion extension penalty per base (in addition to scoreInsOpen).--scoreStitchSJshift: Set to 1. Maximum score reduction while searching for SJ boundaries in the stitching step.--runThreadN: Set to 10. Number of threads used per jobs.
Note : STAR include numerous parameters, please read the manual for more informations.
Varscan; SNP calling¶
AllMine use Varscan v2.4.3 to perform SNP calling on aligned reads.
Parameters :
--p-value: Set to 0.99. We did not choosed to use p value as filter for SNP calling. However you can tune this parameter if wanted.--min-coverage: Set to 8. Minimal deep at SNP loci for calling.--min-var-freq: Set to 0.15. Minimal variant frequency at SNP loci for calling.--min-avg-qual: Set to 20. Minimal sequencing quality at SNP loci for calling.
Note : The tunning of Varscan parameters is a trade-off between sensitivity and specificity. It should be keeped in mind when analysing your results!