Qtip
Background
Read alignment is the first step in most sequencing data analyses. It is also a source of errors and interpretability problems. Repetitive genomes, algorithmic shortcuts, and genetic variation impede the aligner's ability to find a read's true point of origin. Aligners therefore report a mapping quality: the probability the reported point of origin for a read is incorrect.
Qtip is an accurate, aligner-agnostic tool for predicting mapping qualities that works by simulating a set of tandem reads, similar to the input reads in important ways, but for which the true point of origin is known. Alignments of tandem reads are used to build a model for predicting mapping quality, which is then applied to the input-read alignments. The model is automatically tailored to the alignment scenario at hand, allowing it to make accurate mapping-quality predictions across a range of read lengths, alignment parameters, genomes, and read aligners.
Dependencies
- Python 2.7 or later
- Numpy
- Scikit-learn
- Pandas
Building Qtip
If Qtip was cloned/extracted to a directory $QTIP_HOME, then:
make -C $QTIP_HOME/srcUsing Qtip
Qtip runs alongside an existing aligner, though the aligner requires modifications for Qtip to obtain the feature data it needs to make predictions. We have already made these modifications for the popular Bowtie 2, BWA-MEM and SNAP tools. See the software subdirectory for details.
To run qtip, specify input reads, aligner, aligner arguments, genome index, and genome FASTA file.
usage: qtip [-h] --ref path [path ...] [--U path [path ...]]
[--m1 path [path ...]] [--m2 path [path ...]] [--index path]
[--seed int] [--max-allowed-fraglen int] [--input-model-size int]
[--sim-unp-min int] [--sim-conc-min int] [--sim-disc-min int]
[--sim-bad-end-min int] [--sim-function linear|sqrt]
[--sim-factor fraction] [--wiggle int] [--bt2-exe path]
[--bwa-exe path] [--snap-exe path] [--aligner name]
[--write-orig-mapq] [--write-precise-mapq] [--orig-mapq-flag XX:X]
[--precise-mapq-flag XX:X] [--keep-ztz] [--model-family family]
[--num-trees int,int,...] [--max-features float,float,...]
[--max-leaf-nodes int,int,...] [--learning-rate float,float,...]
[--optimization-tolerance fraction] [--reweight-ratio float]
[--reweight-mapq] [--reweight-mapq-offset float] [--collapse]
[--max-rows int] [--no-oob] [--skip-rewrite] [--profile-memory]
[--predict-for-training] [--try-include-mapq]
[--subsampling-series floats] [--trials int] [--assess-accuracy]
[--assess-limit int] [--temp-directory path]
[--output-directory path] [--vanilla-output path]
[--keep-intermediates] [--profile] [--verbose] [--version]
Align a collection of input reads, simulate a tandemdataset, align the tandem
dataset, and emit both theinput read alignments and the training data derived
fromthe tandem read alignments.
optional arguments:
-h, --help show this help message and exit
--ref path [path ...]
FASTA file, or many FASTAs separated by spaces,
containing reference genome sequences (default: None)
--U path [path ...] Unpaired read FASTQ file name, or many FASTQ file
names separated by spaces (default: None)
--m1 path [path ...] Mate 1 FASTQ file name, or many FASTQ file names
separated by spaces; must be specified in same order
as --m2 (default: None)
--m2 path [path ...] Mate 2 FASTQ file name, or many FASTQ file names
separated by spaces; must be specified in same order
as --m1 (default: None)
--index path Index file to use; specify the appropriate prefix,
e.g. Bowtie 2 index file name without the .X.bt2
suffix. (default: None)
--seed int Integer to initialize pseudo-random generator
(default: 99099)
--max-allowed-fraglen int
When simulating fragments, longer fragments are
truncated to this length (default: 100000)
--input-model-size int
Maximum # templates to keep when building an input
model. There are 4 separate models for each alignment
category and this governs the maximum for all 4.
(default: 30000)
--sim-unp-min int If predictions for unpaired reads are needed, simulate
at least this # of unpaired reads. (default: 30000)
--sim-conc-min int If predictions for concordantly aligned reads are
needed, simulate at least this # of concordant pairs.
(default: 30000)
--sim-disc-min int If predictions for discordantly aligned reads are
needed, simulate at least this # of discordant pairs.
(default: 10000)
--sim-bad-end-min int
If predictions for ends with an unaligned mate are
needed, simulate at least this # of pairs with a bad
end. (default: 10000)
--sim-function linear|sqrt
Function giving # of tandem reads to simulate in a
category; parameter is the number of input reads. See
also: --sim-factor. (default: sqrt)
--sim-factor fraction
This is multiplied with X (if --sim-function=linear)
or sqrt(X) (if --sim-function=sqrt) to calculate
number of tandem reads to simulate in a given
category, where X is # of input reads in that
category. (default: 45.0)
--wiggle int Wiggle room to allow in starting position when
determining whether alignment is correct (default: 30)
--bt2-exe path Path to Bowtie 2 aligner exe, "bowtie2" (default:
None)
--bwa-exe path Path to BWA-MEM aligner exe, "bwa" (default: None)
--snap-exe path Path to SNAP aligner exe, "snap-aligner" (default:
None)
--aligner name Which aligner to use: bowtie2 | bwa-mem | snap
(default: bowtie2)
--write-orig-mapq Write original MAPQ as an extra field in output SAM
(default: False)
--write-precise-mapq Write a more precise MAPQ prediction as an extra field
in output SAM (default: False)
--orig-mapq-flag XX:X
If --write-orig-mapq is specified, store original MAPQ
in this extra SAM field (default: Zm:Z)
--precise-mapq-flag XX:X
If --write-precise-mapq is specified, store original
MAPQ in this extra SAM field (default: Zp:Z)
--keep-ztz Don't remove ZT:Z field, with aligner-reported feature
data, from the final output SAM (default: False)
--model-family family
{RandomForest | ExtraTrees | GradientBoosting}
(default: RandomForest)
--num-trees int,int,...
number of decision trees to try; relevant for all
model families (default: 30)
--max-features float,float,...
maximum number of features to consider at each
decision tree node; relevant for RandomForest and
ExtraTrees (default: 0.1,0.2,0.25,0.3,0.35,0.4,0.45)
--max-leaf-nodes int,int,...
maximum number of leaf nodes to include in a decision
tree; relevant for RandomForest and ExtraTrees
(default: 35)
--learning-rate float,float,...
learning rate to use when fitting; only relevant for
GradientBoosting (default: 0.75,0.8,0.85,0.9,0.95,1.0)
--optimization-tolerance fraction
When using hill climbing procedure to optimize
hyperparamters,stop when OOB score can't be improved
by this relative factor (default: 0.01)
--reweight-ratio float
When fitting, reweigh samples so weight of highest-
mapq alignment has this times as much weight as
lowest-mapq. (default: 1.0)
--reweight-mapq When fitting, reweigh samples according to initially-
predicted mapq. Higher predictions get more weight
(default: False)
--reweight-mapq-offset float
Add this to every MAPQ before reweighting (default:
10.0)
--collapse Remove redundant rows just before prediction. Usually
not a net win. (default: False)
--max-rows int Maximum number of rows (alignments) to feed at once to
the prediction function (default: 250000)
--no-oob Don't use out-of-bag score when fitting
hyperparameters -- use cross validation instead. No
effect for models that don't calculate OOB score.
(default: False)
--skip-rewrite Skip the final SAM rewriting step; other results,
including any fit and prediction assessments
requested, are still written. (default: False)
--profile-memory Use guppy/heapy to profile memory and periodically
print heap usage (default: False)
--predict-for-training
Make predictions and produce associated plots/output
files for training (tandem) data (default: False)
--try-include-mapq Try both with and without including the tool-predicted
MAPQ as a feature; default: does not include it
(default: False)
--subsampling-series floats
Comma separated list of subsampling fractions to try
(default: 1.0)
--trials int Number of times to repeat fitting/prediction (default:
1)
--assess-accuracy When correctness can be inferred from simulated read
names, assess accuracy of old and new MAPQ predictions
(default: False)
--assess-limit int The maximum number of alignments to assess when
assessing accuracy (default: 100000000)
--temp-directory path
Write temporary files to this directory; when None:
uses environment variables like TMPDIR, TEMP, etc
(default: None)
--output-directory path
Write outputs to this directory (default: None)
--vanilla-output path
Only write final SAM file; suppress all other output
(default: None)
--keep-intermediates Keep intermediates in output directory; if False,
intermediates are written to a temporary directory
then deleted (default: False)
--profile Print profiling info (default: False)
--verbose Be talkative (default: False)
--version Print version and quit (default: False)Testing Qtip
make -C $QTIP_HOME/testQtip architecture
- Input reads are aligned to the reference genome using the specified aligner and parameters.
- SAM alignments are parsed and an input model, capturing information about the input reads and their alignments, is built.
- Input model and reference genome are used to simulate a new set of reads, called tandem reads since they originate from tandem simulation. Each tandem read is from a random location in the genome and is labeled with its true point of origin.
- Tandem reads are aligned to the reference genome using the same aligner and parameters as in step 1.
- Alignments produced in step 4 are parsed and converted to training records. Because the true point of origin is known, each training record can be labeled as correct or incorrect.
- A model is trained on the records from step 5
- SAM alignments from step 1 are parsed. For each aligned read, a test record, like the training record from step 5, is constructed. Based on the test record, the trained model is applied to predict mapping quality. The alignment's SAM record is then re-written substituting the prediction in the MAPQ field.