概述¶

适用范围和特性¶

pRESTO执行在比对到种系参考序列前的所有阶段的序列处理工作。该工具箱容易使用，但要求对于命令行程序有一定的了解。与针对少数常见的工作流程提供一个固定的解决方案不同的是，我们把 pRESTO设计的尽可能的灵活。这个设计理念使得pRESTO适合于许多现有的protocol，也适用于未来的技术，但是这要求用户去构建一系列命令行和参数以针对他们实验的protocol。

pRESTO由一套独立的工具组成，它们处理具体的任务，通常有一系列不同行为的子命令。下表展示了对每个工具的简要描述。

工具	子命令	描述
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
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.
	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
	sort	Sorts sequences based on annotations

输入和输出¶

所有的工具都以标准FASTA或FASTQ格式文件为输入，并输出同样格式的文件。这使得pRESTO可以和其他使用其中任何一种格式的序列分析工具无缝衔接，如果需要的话，在pRESTO工作流程中的任何步骤都可以被其他工具替代。

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.

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