Instructions

This page allows the user to synthesize oligonucleotides for MPRAs experiments to investigate the rules that govern transcription factor occupancy. A set of variables provides fine control of how motifs are placed into the sequences. The user is able to specify the locations to substitute the motifs, insert restriction sites , adapter sites and barcodes.

Parameters

Enter FASTA sequences

The Fasta sequences as text.

Upload FASTA sequences

The Fasta sequences as a text file.

Enter your motifs

The motifs in FASTA format.

Reverse complement sequence

The option to reverse complement the sequences before motif substitution.

Minimum Spacing

The minimum spacing between the motifs.

Maximum Spacing

The maximum spacing between the motifs.

Distance from left edge

This is the minimum distance from left-most nucleotide of the motif to the left edge of the sequence.

Distance from right edge

This is the minimum distance from right-most nucleotide of the motif to the right edge of the sequence.

Interval of substitution of motifs

This specifies the interval length to insert the motifs

Barcode length

The size of the barcode specified

Minimum barcode GC content (%)

The minimum amount (in percent) of GC content in each of the barcodes

Maximum barcode GC content (%)

The maximum amount (in percent) of GC content in each of the barcodes

Barcode edit distance

The Levenshtein distance between each barcode. The default is 2.

Number of barcodes per sequence

This specifies the number of barcodes inserted per sequence (replicates).

Restriction sites

The ability to add upto 2 restriction sites into the final product.

Adapter sites

The ability to add upto 2 adapter sites into the final product.

Ordering

Allows the user to order the constituent parts therefore providing flexibility in the design of the experiment.

Result page

The result page displays the synthesized oligonucleotides in the FASTA format. The user is able to download (plain text file) the generated oligonucleotides. The description line (header) has information about the options chosen by the user during submission. A header is composed of one or more DESCRIPTORs and each DESCRIPTOR is composed of a LABEL and INFO. The descriptors are delimited by a |, i.e. a "pipe".

Image below is an excerpt of the Results page ( showing 8 nucleotides ). The nucleotides in red are the substituted motifs.

The format of the FASTA header for each sequence is shown below. Note that the order of the DESCRIPTOR in the header is not significant.

> <LABEL> - <INFO> | <LABEL> - <INFO> | <LABEL> - <INFO> | ...

The LABEL is one of the options as shown below and INFO is either a number or a word which describes the LABEL in more detail.

Label types:

<MOTIF>

A particular motif inserted by the user.

BARCODE

The variant of the same sequence

RESTRICTION

This is the restriction site put by the user

ADAPTER

The presence of the adapter sequence of the specified number

DUPLICATE_RESTRICTION_SITES

The restriction site which has multiple copies present.

Example of a header:

> ATGTG - 53|AAAAA-61|RESTRICTION - 1|RESTRICTION - 2

There are 4 DESCRIPTORs.

ATGTG

53 is the motif starting at position 54 in the background sequence.

AAAAA

61 is the motif starting at position 61 in the background sequence.

RESTRICTION

1 signifies the presence of the restriction site 1 in the final sequence.

RESTRICTION

2 signifies the presence of the restriction site 2 in the final sequence.

This sequence neither has a barcode nor any adapter sites. There are also no spurious presence of any restriction site.