Classifications

• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
  • G16B30/20—Sequence assembly

Definitions

• This invention is generally related to the field of molecular biology, and more specifically the field of primer design for targeted/high-throughput sequencing.
• Primer designing for DNA sequencing is a vital part for modern biological research.
• Traditional primer designing programs for example publically available
• Primer3 use a single input DNA sequence to process and design optimal primers under specified parameters including primer length, GC content, melting temperature (Tm) and others.
• Tm melting temperature
• these traditional primer designing programs are not suitable for handling large files with long or multiple sequences.
• the target region can be a small ( ⁇ 1 kilo bases or kb) to large contiguous or repeat-masked regions (even entire genomes provided sufficient hardware and memory to handle the processes).
• the systems and methods provided herein can be used to design multiple sets of overlapping or non-overlapping primers/amplicons for a target region.
• the primers designed using the systems and methods provided herein can also be used for multiplex PCR analysis.
• a computerized system for primer/amplicon design for sequencing comprises:
• the system further comprises at least one of format output module, and a BLAST verification module. In a further embodiment, the system further comprises at least one of format output module, BLAST verification module, and adaptor verification module.
• the input device is selected from the group consisting of automated sequencer, sequencing data input device, and sequencing data storage device.
• the output interface comprises interface for
• the database described herein contains information selected from the group consisting of genomic sequences, previously generated primers, and sequences for BLAST analysis.
• the load sequence module processes sequences in FASTA format.
• the load sequence module uses random file access.
• the load sequence module does not use sequential file access.
• the primer design module performs at least one of
• the primer design module processes primer design in parallel. In another embodiment, the primer design module does not design primers in a non-parallel or sequential manner. In another embodiment, the primer design module generates primers or processes primer design at a speed greater than 10 primers per minute. In another embodiment, the primer design module generates primers or processes primer design at a speed greater than 100 primers per minute.
• the primer design module generates primers or processes primer design at a speed between 200 and 500 primers per minute.
• the primers constitute overlapping amplicons for sequence assembly.
• the primers constitute overlapping amplicons for sequencing and assembly.
• the overlapping region of amplicons comprises at least 50 bp or minimal overlap.
• the overlapping region of amplicons comprises at least 100 bp.
• the overlapping region of amplicons comprises between 100 bp and 1000 bp.
• a method for use in a computerized system for primer/amplicon design for sequencing comprises:
• the method further comprises pre-processing sequences by modifying sequences before primer design.
• the method further comprises defining target regions/sequences.
• the method further comprises defining windows for primer design.
• the computerized system of the method comprises a system described herein.
• the sequence data is larger than 100 kilo bases (kb).
• the sequence data is larger than 10 Mega bases (mb).
• the sequence data is between 10 mb and 1 giga bases (gb).
• the load sequence module processes sequences in FASTA format.
• the load sequence module uses random file access.
• the method provided further comprises at least one of (1) automatically adding standard 5' tag or tail to each primer; (2) selecting nested primer pairs; (3) selecting primers for multiplexed amplifications; (4) designing a tiling of amplicons across a sequence; (5) picking primers from a reverse-translated amino acid sequence; and (6) selection from multiple primer sets.
• the method provides primers at a speed greater than 10 primers per minute. In a further or alternative embodiment, the method provides primers at a speed greater than 100 primers per minute. In a further or alternative embodiment, the method provides primers at a speed between 200 and 500 primers per minute. In another embodiment, the primers constitute overlapping amplicons for sequence assembly. In a further embodiment, the overlapping region of amplicons comprises at least 50 bp or minimal overlap. In a further embodiment, the overlapping region of amplicons comprises at least 100 bp. In a further embodiment, the overlapping region of amplicons comprises between 100 bp and 1000 bp.
• the method further comprises verifying the primers using a BLAST verification module. In another embodiment, the method further comprises verifying secondary structure of primers using an adaptor verification module. In another embodiment, the method further comprises simulating the sequencing using a sequencing simulation module. In another embodiment, the method further comprises an output format module for outputting for visualization using WebGBrowse. BRIEF DESCRIPTION OF THE DRAWINGS
• Figure 1 shows an exemplary flowchart of the Primer Designing Pipeline provided herein.
• Figure 2 shows an exemplary embodiment for overlapping amplicon design for high-throughput sequencing.
• Figure 3 shows an exemplary automated process for the systems and methods provide herein.
• Figure 4 shows an exemplary process for the BLAST verification modules and methods provided herein.
• Figure 5 shows exemplary FASTA sequences to be loaded into the Primer Designing Pipeline provided herein.
• Figure 6 shows an exemplary screen shot when the Primer Designing Pipeline provided loads FASTA files for downstream analysis.
• a publically available "Primer3" program is incorporated by the systems and methods provided to process the overlapping primer designing task in targeted regions while also combining the utility of Batch-Primer3 using a customized and compiled program.
• the "Primer3" program has been previously described in Steve Rozen and Helen J.
• the Primer Designing Pipeline provided is programmed using .NET framework and allows multiple "Primer3" processes to be performed in parallel while validating for overlap.
• the Primer Designing Pipeline described herein provides at least one of the advantages below: (1) automatically adding standard 5' tag or tail to each primer; (2) selecting nested primer pairs; (3) selecting primers for multiplex amplifications; (4) designing a tiling of amplicons across a sequence; and (5) picking primers from a reverse-translated amino acid sequence.
• the systems and methods provided herein enable primer designing especially for "targeted re- sequencing” applications, for example, using high- throughput (HTP) next-generation sequencing (NGS) instruments.
• the Primer Designing Pipeline provided can be modular to take small to large sequences as input and also allows changes of the amplicon lengths to suit various NGS platforms as requested by users.
• primers designed using the systems and/or methods provided herein can be used with Fluidigm AccessArray system, a HTP multiplexed amplicon library generation system for efficient and cost-effective generation of sequencing data for further analysis.
• the systems and methods described herein can provide HTP overlapping primer design for complementing the utility of Fluidigm AccessArray system for marker development, gene confirmation, transgene region validation for regulatory affairs, QTL mining and genotyping-by-sequencing.
• FIG. 1 An exemplary primer design workflow is illustrated in Figure 1.
• the users can select sequence files for which they want to design primers. Files are typically not loaded into memory but instead analyzed for random access.
• the FASTA file format is a common platform for displaying biological sequences.
• the target sequence(s) can typically be provided in a FASTA format.
• the FASTA file can contain one or multiple sequences.
• Each sequence is always preceded by a header line which is prefixed with ">" followed by the ID, description, and/or other pertinent information about the sequence.
• the sequence information is then listed on subsequent lines and usually wraps (carriage return/line feed) every 70 or 80 characters depending on the program that generated the file.
• wraps carriage return/line feed
• DNA sequences in FASTA format are shown in Figure 5.
• FASTA files can become quite large when dealing with long sequences, a large number sequences, or a combination of both.
• most FASTA files which represent the entire genome of complex eukaryotes may easily exceed 2 gigabytes.
• This size issue poses a problem for the primer design process because normal file handling methods involve starting at the beginning and reading the file into memory until the data of interest is found. For example, a sequential file access for a file larger than 2 gigabytes may take longer than 30 seconds to sequentially read to the spot in the file where the data of interest is. Consequently, this process has to take place for each set of primers which need to be designed for traditional primer designing programs.
• a Load Sequence Module which runs the sequence loading processes in parallel. Random file access is combined with sequential file access to speed up the process. Each character in the file resides at a specific addressable location on a disk. In addition to starting at the beginning of the file and reading each character in order (sequential file access), the Load Sequence Module provides a means to access any location in the file at random as long as the address is known. Random file access can speed processes up considerably because the process does not have to read all the characters/data that came before the data in the file that it's interesting in extracting. Instead of being obligated to perform sequential file access faster, the Load Sequence Module provided is able to determine the starting address in the file at which the data of interest is located.
• the Primer Designing Pipeline provided initially reads the entire file sequentially through once, analyzes it to determine how it' s formatted, and stores that analysis in a SQL Server Compact database. Then when the Primer Designing Pipeline provided is designing the primers and needs to extract a sequence from one of the files, the Primer Designing Pipeline provided uses the analysis results stored in the SQL Server Compact database to calculate the location within the file (or address) of the data of interest. Thus, that address is used to extract/read the data using random file access.
• each block has the same line length, and it stores information about how many lines, characters per line, and file start and stop position for each block. For example as shown in Figure 5, the first block would start at file position 0, contain 1 line with 13 characters per line (a newline and carriage return exist at the end of the line), and the block would end at position 12. The block after that would start at file position 13, contain 2 lines with 72 characters long, and end at position 156. Additionally, since Load Sequence Module assumes that each header block is unique, each header block can be loaded into a hash table (dictionary) in memory for quick access when working with the file. The blocks following the header which contain the sequence are then linked to the header block in the hash table.
• a hash table dictionary
• the Primer Designing Pipeline provided would look up the header up in the hash table first. Then iterates through each block sequentially to see if the sequence starts in that block. If the file format follows the normal FASTA format, then the Primer Designing Pipeline provided should at most only have to check two blocks because there should be only be two blocks for each sequence in the file. Once the block containing the starting position is determined, the position within that block can be calculated because each character takes up one position/byte in the file.
• each block is also analyzed for what type of newline characters as well as other whitespace characters occur at the end each line in the block. For example, if the following target "SEQUENCE_2 I Corn Sequence Gene A45: 136,8" (header : start, length) is needed to be extracted, then the Primer Designing Pipeline provided would determine that it fell in the first block following the header. For example, the block starts at file position 222, contains 3 lines with 72 characters per line, and there are 2 ending whitespace characters in each line.
• Figure 6 shows an exemplary screenshot illustrating the part of the Load Sequence Module used to load the example FASTA file as shown in Figure 5, where the first block of each section is the header block.
• Targets step segments for which primers to be designed are define by the user(s) (for example all sequences or only masked regions greater than a specified length) or loaded from a file such as a GFF file.
• the GFF format is useful as input files for programs like WebGBrowse, which is previously disclosed in Ram Podicheti, Rajesh Gollapudi, and Qunfeng Dong. (2009) "WebGBrowse - a web server for GBrowse.” Bioinformatics, 25(12): 1550-1551, the content of which is incorporated by reference in its entirety.
• Step 1 is the Load / Define Window step, where the area in which primers to be placed is defined (for example 100 bps up and downstream from the target) or loaded from a file.
• the next step is Enter Primer Parameters, where parameters including primer length, melting temperature, GC content, 3' stability, and/or estimated secondary structure.
• Primer3 is used as the primary design engine and use(s) can enter parameters as required by the Primer3 program.
• a Primer Design Module having two major functions: (1) selecting and processing the target and (2) saving the results.
• the algorithm of the Primer Design Module may adjust settings internally. For example with HTP primer design, the Primer Design Module can start at the beginning of the target area and design overlapping primer sets until it reaches the end. In one embodiment, additional evaluation may be needed such as BLASTing or secondary structure prediction before moving to the next set of primers.
• Basic Local Alignment Search Tool (BLAST) is a commonly used sequence alignment tool. See Altschul et al. (1990) /. Mol. Biol. 215: 403- 410, the content of which is hereby incorporated by reference in its entirety.
• the Primer Designing Pipeline provided automatically generates and adds specified adaptor sequences to the designed primers.
• the user(s) When performing targeted genome sequencing where only specific sub- sequences within a genome are desired, the user(s) must manually design primers to create overlapping amplicons where the targeted region is larger than the maximum read length of the sequencing equipment. To date, most high throughput sequencing machines can only sequentially read a limited number of base pairs in one run. Thus, the source genetic material needs to be chopped up into segments that are less than the maximum read length. In order to assembly these segments back into one sequence, the segments need to have some overlap sequence (usually at least 20 base pairs).
• Figure 2 shows an exemplary embodiment of high-throughput sequencing where a sequencer is used to sequence the target region.
• the target region is 5,000 base pairs long and the sequencer can only read segments of DNA up to 700 base pairs - i.e., the 5,000 bp sequence needs to be "chopped" in 700 bp (base pairs) or less segments.
• the source sequence isn't chopped but the 5,000 bp sequence is amplified into 700 bp segments for sequencing.
• shorter overlapping copies are made instead.
• the reads are stored in a data file and an assembly program assembles them back into one continuous sequence.
• primers In order to make the shorter copies or amplicons during the amplification stage of the sequencing, primers, short sequences that mark the beginning and end of an amplicon, have to be designed and created.
• Traditional tools for designing primers can only design one set of primers (or one amplicon) at a time. These overlapping amplicons have to be designed serially (one after the other) and usually in a fairly manual process.
• the automated systems and methods for designing primers for overlapping amplicons.
• the automated systems and methods start from a traditional primer design program/software, for example Primer3, which can be downloaded locally and run from a command line interface from either a Linux or Windows machine.
• the automated systems and methods provided are generated using Perl script (parallelized or non-parallelized).
• the automated systems and methods provided are generated using Microsoft .NET 4.0. , which contains functionality for parallelizing processes.
• the systems and methods provided using Microsoft .NET 4.0 can design large batches of primers in a few minutes as compared to hours using non-parallelized Perl script or days with the traditional approaches.
• the automated systems and methods provided use a parallelized approach.
• the automated systems and methods provided does not use a non-parallelized approach.
• Figure 3 shows an exemplary system provided using Microsoft .NET 4.0.
• the output from the .NET program is a tab delimited test file containing the primer sequences and information about the quality of the primers.
• the Primer Designing Pipeline provides also saves copies of the input sent to Primer3 and the output Primer3 generates. This can be useful in troubleshooting any issues that may arise or manually re-running one portion of the process if necessary.
• a Format Output Module which reads the output from the automation program and generate a general feature file (GFF) formatted file that can be used by other programs including GBrowse to visually overlay the primers and amplicons on the source sequence.
• GFF general feature file
• the raw results from the Primer Designing Pipeline are compiled and formatted into a tab delimited format as well as optionally a GFF format for feeding into GBrowse for visualization. For one example, 14 pairs of
• primers/amplicons are created within four minutes using the systems and methods provided. These 14 pairs of primers form overlapping amplicons over one broad targeted sequence. For another example, 9 pairs of primers/amplicons (i.e., 18 primers) are created within two minutes using the systems and methods provided. These 9 pairs of primers from overlapping amplicons over two separated targeted sequences, where the two targeted sequences are still within the same genome (i.e., skipping one region in between for sequencing).
• the systems and/or methods provided also comprise a BLAST Verification Module.
• An exemplary BLAST Verification Module is illustrated in Figure 4.
• the BLAST Verification Module verifies target redundancy for amplicon primer design.
• the BLAST Verification Module will take the outputs and BLAST them against the targeted genome or sequence library available in database.
• the BLAST Verification Module allows a primer set (pair) or amplicon to be unique based on BLAST analysis.
• the BLAST Verification Module provides BLAST analysis in parallel, thus saving time as compared to sequential analysis.
• primers from the first set typically have to be BLASTed then the next one, and then the results from both BLAST queries must be compared to see if both primers land within pre-determined number of base pairs from each other (usually 1000) and are on opposite strands of the DNA pointing the correct direction for amplification to occur. If non-unique primers are found, then those specific sequences need to be re-run through the primer design process with different parameters.
• the BLAST Verification Module specifies a BLAST database to use before running the primer design process. As the primer design got back individual results, the Primer Designing Pipeline disclosed can automatically check them for uniqueness and try to re-run that sequence if necessary.
• a copy of the BLAST database is created locally on the user's workstation. In other embodiments, the BLAST databases is located on a serve and accessed remotely by the Primer Designing Pipeline.
• the systems and/or methods provided also comprise an Adaptor Verification Module.
• the Primer Designing Pipeline adds adapter/tag sequences to the primers in order for the sequencing machine to be able to sequence the amplicon. This adapter/tag sequence is often the same for all primers.
• the secondary structure of a designed primer may change significantly after adding such adaptor/tag sequence.
• RNAstructure for both DNA and RNA
• RNA has been developed by the University of Rochester that can be used to predict the most likely binding structure a sequence or pair sequences can make.
• the Adaptor Verification Module of the Primer Designing Pipeline can automate the RNAstructure program through the command line and enable prediction whether after adding an adapter sequence, a primer set is still a scientifically good choice.
• the Adaptor Verification Module comprises an internal scoring system for classifying primers based on the predicted secondary structure.
• Other programs can be used for the Adaptor Verification Module provided including Mfold (as disclosed in M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-3415, 2003) and UNAFold (as disclosed in N. R. Markham & M. Zuker. UNAFold: Software for Nucleic Acid Folding and Hybridization. In Data, Sequence Analysis, and Evolution, J. Keith, ed., Bioinformatics: Volume 2, Chapter 1, pp 3-31, Humana Press Inc., 2008), the content of both are hereby incorporated by reference in their entireties.
• simulation of amplification and/or sequencing can be performed.
• Several programs have been disclosed to simulate the entire sequencing process, for example in silico PCR amplification for amplification, MetaSim for simulating sequencing, and CAP3 for assembly.
• the Primer Designing Pipeline can integrate the ability to simulate the entire sequencing process using a series of simulators.

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