Overview

Scope and Features

pRESTO performs all stages of raw sequence processing prior to alignment against reference germline sequences. The toolkit is intended to be easy to use, but some familiarity with commandline applications is expected. Rather than providing a fixed solution to a small number of common workflows, we have designed pRESTO to be as flexible as possible. This design philosophy makes pRESTO suitable for many existing protocols and adaptable to future technologies, but requires users to construct a sequence of commands and options specific to their experimental protocol.

pRESTO is composed of a set of standalone tools to perform specific tasks, often with a series of subcommands providing different behaviors. A brief description of each tool is shown in the table below.

Tool Subcommand Description
AlignSets   Multiple aligns sets of sequences sharing the same annotation
  muscle Uses the program MUSCLE to align reads
  offset Uses a table of primer alignments to align the 5’ region
  table Creates a table of primer alignments for the offset subcommand
AssemblePairs   Assembles paired-end reads into a complete sequence
  align Assembles paired-end reads by aligning the sequence ends
  join Concatenates pair-end reads with intervening gaps
  reference Assembles paired-end reads using V-segment references
  sequential Attempt alignment assembly followed by reference assembly
BuildConsensus   Constructs UMI consensus sequences
ClusterSets   Clusters UMI read groups into smaller sub-groups
CollapseSeq   Removes duplicate sequences
ConvertHeaders   Converts sequence headers to the pRESTO format
  generic Converts sequence headers with an unknown annotation system
  454 Converts Roche 454 sequence headers
  genbank Converts NCBI GenBank and RefSeq sequence headers
  illumina Converts Illumina sequence headers
  imgt Converts sequence headers output by IMGT/GENE-DB.
  migec Converts sequence headers output by MIGEC.
  sra Converts NCBI SRA sequence headers
EstimateError   Estimates error rates for UMI data
FilterSeq   Removes or modifies low quality reads
  length Removes sequences under a defined length
  maskqual Masks low Phred quality score positions with Ns
  missing Removes sequences with a high number of Ns
  repeats Removes sequences with long repeats of a single nucleotide
  quality Removes sequences with low Phred quality scores
  trimqual Trims sequences to segments with high Phred quality scores
MaskPrimers   Identifies and removes primer regions, MIDs and UMI barcodes
  align Matches primers by local alignment and reorients sequences
  score Matches primers at a fixed user-defined start position
PairSeq   Sorts paired-end reads and copies annotations between them
ParseHeaders   Manipulates sequence annotations
  add Adds a field and value annotation pair to all reads
  collapse Compresses a set of annotation fields into a single field
  copy Copies values between annotations fields
  delete Deletes an annotation from all reads
  expand Expands an field with multiple values into separate annotations
  rename Rename annotation fields
  table Outputs sequence annotations as a data table
ParseLog   Converts the log output of pRESTO scripts into data tables
SplitSeq   Performs conversion, sorting, and subsetting of sequence files
  count Splits files into smaller files
  group Splits files based on numerical or categorical annotation
  sample Randomly samples sequences from a file
  samplepair Randomly samples paired-end reads from two files
  select Filters sequences based on annotations
  sort Sorts sequences based on annotations

Input and Output

All tools take as input standard FASTA or FASTQ formatted files and output files in the same formats. This allows pRESTO to work seamlessly with other sequence processing tools that use either of these data formats; any steps within a pRESTO workflow can be exchanged for an alternate tool, if desired.

Each tool appends a specific suffix to its output files describing the step and output. For example, MaskPrimers will append _primers-pass to the output file containing successfully aligned sequences and _primers-fail to the file containing unaligned sequences.

See also

Details regarding the suffixes used by pRESTO tools can be found in the Commandline Usage documentation for each tool.

Annotation Scheme

The majority of pRESTO tools manipulate and add sequences-specific annotations as part of their processing functions using the scheme shown below. Each annotation is delimited using a reserved character (| by default), with the annotation field name and values separated by a second reserved character (= by default), and each value within a field is separated by a third reserved character (, by default). These annotations follow the sequence identifier, which itself immediately follows the > (FASTA) or @ (FASTQ) symbol denoting the beginning of a new sequence entry. The sequence identifier is given the reserved field name ID. To mitigate potential analysis errors, each tool in pRESTO annotates sequences by appending values to existing annotation fields when they exist, and will not overwrite or delete annotations unless explicitly performed using the ParseHeaders tool. All reserved characters can be redefined using the command line options.

FASTA Annotation
>SEQUENCE_ID|PRIMER=IgHV-6,IgHC-M|BARCODE=DAY7|DUPCOUNT=8
NNNNCCACGATTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTTTCTACCAAAA
FASTQ Annotation
@SEQUENCE_ID|PRIMER=IgHV-6,IgHC-M|BARCODE=DAY7|DUPCOUNT=8
NNNNCCACGATTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTTTCTACCAAAA
+
!!!!nmoomllmlooj\Xlnngookkikloommononnoonnomnnlomononoojlmmkiklonooooooooomoo

See also

  • Details regarding the annotations added by pRESTO tools can be found in the Commandline Usage documentation for each tool.
  • The ParseHeaders tool provides a number of options for manipulating annotations in the pRESTO format.
  • The ConvertHeaders tool allows you convert several common annotation schemes into the pRESTO annotation format.