Specific comments
In the following you can find some additional information for more specific applications or software
Paired-end
In paired-end mode, both reads are used for the classification. Read pairs with conflicting reads (tag CF) or pairs containing both tags G1 and G2 are considered conflicting and are not reported by default. Reporting of these reads can be enabled using the option --conflicting.
Singleton alignments in the allele-tagged paired-end file (which is the default for e.g. TopHat) are also sorted into the above four files. Specifying --singletons will write these alignments to special singleton files instead (ending in *_st.bam).
Hi-C
Assumes data processed with HiCUP as input, i.e. the input BAM files are by definition paired-end and Reads 1 and 2 follow each other. Hi-C sorting discriminates several more possible read combinations:
Again, read pairs containing a conflicting reads (tag XX:Z:CF) are not printed out by default, but this may be enabled using the option --conflicting. For an example report please see below.
STAR
RNA-seq alignment files produced by the Spliced Transcripts Alignment to a Reference (STAR) aligner also work well with SNPsplit, however a few steps need to be adhered to make this work:
1) Since SNPsplit only recognises the CIGAR operations M, I, D and N (see above), alignments need to be run in end-to-end mode and not using local alignments (which may result in soft-clipping). This can be accomplished using the option: --alignEndsType EndToEnd
Edit 29 09 2020: As of version 0.4.0, SNPsplit also supports soft-clipped reads (CIGAR operation: S).
2) SNPsplit requires the MD:Z: field of the BAM alignment to work out mismatches involving masked N positions. Since STAR doesn’t report the MD:Z: field by default it needs to be instructed to do so, e.g.: --outSAMattributes NH HI NM MD
3) To save some time and avoid having to sort the reads by name, STAR can be told to leave R1 and R2 following each other in the BAM file using the option: --outSAMtype BAM Unsorted
HISAT2
DNA or RNA alignment files produced by HISAT2 also work well with SNPsplit if you make sure that HISAT2 doesn’t perform soft-clipping. At the time of writing the current version of HISAT2 (2.0.3-beta) does perform soft-clipping (CIGAR operation: S) even though this is not well documented (or in fact the documentation on Github suggests that the default mode is end-to-end which should not perform any soft-clipping whatsoever). Until the end-to-end mode works as expected users will have to set the penalty for soft-clipping so high that it is effectively not performed (--sp is the option governing the soft-clipping penalty). We suggest adding the following option to the HISAT2 command: --sp 1000,1000
Edit: HISAT2 does now also have an option --no-softclip which should have the same effect.
Edit 29 09 2020: As of version 0.4.0, SNPsplit also supports soft-clipped reads. (CIGAR operation: S).
BWA
The other very popular Burrows-Wheeler Aligner (BWA) is unfortunately not compatible with SNPsplit alignment sorting as was disscussed in more detail here. Briefly, the reason for this is that BWA randomly replaces the ambiguity nucleobase N in the reference by either C, A, T or G, thereby rendering an N-masked allele-sorting process impossible.
Bisulfite-seq data
This mode assumes input data has been processed with the bisulfite mapping tool Bismark. SNPsplit will run a quick check at the start of a run to see if the file provided appears to be a Bismark file, and set the flags --bisulfite and/or --paired automatically. In addition it will perform a quick check to see if a paired-end file appears to have been positionally sorted, and if not will set the --no_sort flag (this data is extracted from the @PG header line). Paired-end (--paired) or bisulfite (--bisulfite) mode can also be set manually. Paired-end mode requires Read 1 and Read 2 of a pair to follow each other in consecutive lines. Please be aware that files should not be name-sorted using Sambamba.
Utilisation of SNP positions and allele assignment of bisulfite reads
In contrast to the standard mode of using all known SNP positions, SNPs involving C to T transitions may not be used for allele-specific sorting since they might reflect either a SNP or a methylation state. This includes all of the following Reference/SNP combinations:
C/T or T/C for forward strand alignments, and G/A or A/G for reverse strand alignments.
The number of SNP positions that have been skipped because of this bisulfite ambiguity is reported in the report file. These positions may however be used to assign opposing strand alignments since they do not involve C to T transitions directly. For that reason, the bisulfite call processing also extracts the bisulfite strand information from the alignments in addition to the base call at the position involved. For any SNPs involving C positions that are not C to T SNPs both methylation states, i.e. C and T, are allowed to match the C position.
For SNPs which are masked by Ns in the genome no methylation call will be performed, i.e. they will receive a . (dot) in the methylation call string. This means that SNP positions are used for allele-sorting only but do not participate in calling methylation. While this may reduce the number of total methylation calls somewhat it effectively eliminates the problem of using positions with potentially incorrect methylation status.