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README.md
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sam_insert-size.pl
sam_insert-size.pl
 
 

README.md

sam_insert-size

sam_insert-size.pl is a script to calculate insert size and read length statistics for paired-end reads in SAM/BAM format.

Synopsis

perl sam_insert-size.pl -i file.sam

or

samtools view -h file.bam | perl sam_insert-size.pl -i -

Description

Calculate insert size and read length statistics for paired-end reads in SAM/BAM alignment format. The program gives the arithmetic mean, median, and standard deviation (stdev) among other statistical values.

Insert size is defined as the total length of the original fragment put into sequencing, i.e. the sequenced DNA fragment between the adaptors. The 16-bit FLAG of the SAM/BAM file is used to filter reads (see the SAM specifications).

Read length statistics are calculated for all mapped reads (irrespective of their pairing).

Insert size statistics are calculated only for paired reads. Typically, the insert size is perturbed by artifacts, like chimeras, structural re-arrangements or alignment errors, which result in a very high maximum insert size measure. As a consequence the mean and stdev can be strongly misleading regarding the real distribution. To avoid this, two methods are implemented that first trim the insert size distribution to a 'core' to calculate the respective statistics. Additionally, secondary alignments for multiple mapping reads and supplementary alignments for chimeric reads, as well as insert sizes of zero are not considered (option -min_ins_cutoff is set to one by default).

The -a|-align method includes only proper/concordant paired reads in the statistical calculations (as determined by the mapper and the options for insert size minimum and maximum used for mapping). This is the default method.

The -p|-percentile method first calculates insert size statistics for all read pairs, where the read and the mate are mapped ('raw data'). Subsequently, the 10th and the 90th percentile are discarded to calculate the 10% truncated mean and stdev. Discarding the lowest and highest 10% of insert sizes gives the advantage of robustness (insensitivity to outliers) and higher efficiency in heavy-tailed distributions.

Alternative tools, which are a lot faster, are CollectInsertSizeMetrics from Picard Tools and sam-stats from ea-utils.

Usage

samtools view -h file.bam | perl sam_insert-size.pl -i - -p -d -f -min 50 -max 500 -n 2000000 -xlim_i 350 -xlim_r 200

Options

Mandatory options

  • -i, -input

    Input SAM file or piped STDIN (-) from a BAM file e.g. with samtools view from Samtools

  • -a, -align

    Default method: Align method to calculate insert size statistics, includes only reads which are mapped in a proper/concordant pair (as determined by the mapper). Excludes option -p.

or

  • -p, -percentile

    Percentile method to calculate insert size statistics, includes only read pairs with an insert size within the 10th and the 90th percentile range of all mapped read pairs. However, the frequency distribution as well as the histogram will be plotted with the 'raw' insert size data before percentile filtering. Excludes option -a.

Optional options

  • -h, -help

    Help (perldoc POD)

  • -d, -distro

    Create distribution histograms for the insert sizes and read lengths with R. The calculated median and mean (that are printed to STDOUT) are plotted as vertical lines into the histograms. Use it to control the correctness of the statistical calculations.

  • -f, -frequencies

    Print the frequencies of the insert sizes and read lengths to tab-delimited files 'ins_frequency.txt' and 'read_frequency.txt', respectively.

  • -max, -max_ins_cutoff

    Set a maximal insert size cutoff, all insert sizes above this cutoff will be discarded (doesn't affect read length). With -min and -max you can basically run both methods, by first running the script with -p and then using the 10th and 90th percentile of the 'raw data' as -min and -max for option -a.

  • -min, -min_ins_cutoff

    Set a minimal insert size cutoff [default = 1]

  • -n, -num_read

    Number of reads to sample for the calculations from the start of the SAM/BAM file. Significant statistics can usually be calculated from a fraction of the total SAM/BAM alignment file.

  • -xlim_i, -xlim_ins

    Set an upper limit for the x-axis of the 'R' insert size histogram, overriding automatic truncation of the histogram tail. The default cutoff is one and a half times the third quartile Q3 (75th percentile) value. The minimal cutoff is set to the lowest insert size automatically. Forces option -d.

  • -xlim_r, -xlim_read

    Set an upper limit for the x-axis of the optional 'R' read length histogram. Default value is as in -xlim_i. Forces option -d.

  • -v, -version

    Print version number to STDERR

Output

  • STDOUT

    Calculated stats are printed to STDOUT

  • ./results

    All optional output files are stored in this results folder

  • (./results/ins_frequency.txt)

    Frequencies of insert size 'raw data', tab-delimited

  • (./results/ins_histo.pdf)

    Distribution histogram for the insert size 'raw data'

  • (./results/read_frequency.txt)

    Frequencies of read lengths, tab-delimited

  • (./results/read_histo.pdf)

    Distribution histogram for the read lengths. Not informative if there's no variation in the read lengths.

Run environment

The Perl script runs under Windows and UNIX flavors.

Dependencies

  • Statistics::Descriptive

    Perl module to calculate descriptive statistics, if not installed already get it from CPAN

  • Statistical computing language R

    Rscript is needed to plot the histograms with option -d

Author - contact

Andreas Leimbach (aleimba[at]gmx[dot]de; Microbial Genome Plasticity, Institute of Hygiene, University of Muenster)

Acknowledgements

References/thanks go to:

Citation, installation, and license

For citation, installation, and license information please see the repository main README.md.

Changelog

  • v0.2 (29.10.2014)
    • Fixed bug for options '-min_ins_size' and '-max_ins_size'
    • warn if result files already exist
    • simplify prints to R script with Perl function 'select'
    • minor Perl syntax changes so all Perl scripts conform to the same syntax
    • minor changes to POD
    • finally included README.md
  • v0.1 (27.11.2013)