Standard Read Mapping

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Important notes to the user of this service

Current reference genomes are human (GRCh37) and mouse (mmv37), further genomes will follow, feel free to ask for the genome you are interested in.
To get best mapping results we need information about the used technology (GAII, HiSeq, 454, SOLiD, ...) and preprocessing steps (e.g. use of illumina pipeline). Additionally, a summary of the library preparation steps is helpful.

Required input data

read files (single-end reads or paired-end reads) in Sanger FASTQ format or Illumina FASTQ format (or fastq.gz )

@<SEQ_ID>
	        <SEQUENCE>
	        <+><DESCRIPTION>
                <QUALITY VALUES>

information about technology and preprocessing steps

Generated results

.bam file: sorted mapped alignment file
.bai file: bam index file, necessary for many downstream tools, e.g. IGV
summary report: containing information about total number of reads, number of mapped reads and number of unmapped reads

Additional options on request

removal of duplicates
removal of unmapped reads
coverage plots: graphical overview of the read distribution over the genome
bwa run with non-standard parameters (see standard parameters below)

Methods & Implementation

Mapping of next generation sequence reads against a reference genome is done by Burrows-Wheeler Aligner (bwa) with standard parameters. The output is converted using samtools into .sam format and finally into .bam format.

BWA standard parameters

(This information is subject to change and is taken from here)

ALN

OPTIONS:

-n NUM	Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]
-o INT	Maximum number of gap opens [1]
-e INT	Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]
-d INT	Disallow a long deletion within INT bp towards the 3’-end [16]
-i INT	Disallow an indel within INT bp towards the ends [5]
-l INT	Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ‘-k 2’. [inf]
-k INT	Maximum edit distance in the seed [2]
-t INT	Number of threads (multi-threading mode) [1]
-M INT	Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]
-O INT	Gap open penalty [11]
-E INT	Gap extension penalty [4]
-R INT	Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).
-c	Reverse query but not complement it, which is required for alignment in the color space.
-N	Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.

SAMSE

OPTIONS:

-n INT	Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]
-r STR	Specify the read group in a format like ‘@RG\tID:foo\tSM:bar’. [null]

SAMPE

OPTIONS:

-a INT	Maximum insert size for a read pair to be considered being mapped properly. Since 0.4.5, this option is only used when there are not enough good alignment to infer the distribution of insert sizes. [500]
-o INT	Maximum occurrences of a read for pairing. A read with more occurrneces will be treated as a single-end read. Reducing this parameter helps faster pairing. [100000]
-P	Load the entire FM-index into memory to reduce disk operations (base-space reads only). With this option, at least 1.25N bytes of memory are required, where N is the length of the genome.
-n INT	Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]
-N INT	Maximum number of alignments to output in the XA tag for disconcordant read pairs (excluding singletons). If a read has more than INT hits, the XA tag will not be written. [10]
-r STR	Specify the read group in a format like ‘@RG\tID:foo\tSM:bar’. [null]

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