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Abcloner

Input fasta sequence

Copy and past sequences into the text area below, or upload one or more files containing sequences. Sequences should be in fasta format. For analysis at clone level, the sequence header should comprise clone identifier and sequence identifier that are connected with a hyphen (e.g., EMLK078-HC_A10). Press the 'Example' button at Abcloner page for more details.

Optional primer table

It is optional to upload a spreadsheet containing primers designed for V-genes and J-genes. This table allows Abcloner to assign primer pair to each single-cell clone for PCR amplification purpose. Three columns within the table ('Primer', 'Sequence', and 'V gene segment' ) will be captured by the program. Other information is optional and has no impact on the analysis. An example table is shown below and can be downloaded at the Abcloner application page.


Optional clone table

It is optional to upload a spreadsheet containing clone identifiers and their positions in a plate. The clone identifiers should be identical to those included in sequence header. The numbers of columns and rows are flexible. The first column and row will be treated as header information (see below).

As output, Abcloner maps antibody pairing status for each clone using color code onto the clone table. A pairing status is considered if a functional heavy chain and at least a functional light chains are found for a clone. The color coding is explained in the first column to the right. For each row, the total number of clones with paired status is provided in the second column to the right (see below).


Abgermline

Input fasta sequence

Copy and past sequences into the text area below, or upload a file containing multiple sequences. Sequences should be in fasta format.

Ablineage

Input fasta sequence

Copy and past sequences into the text area below, or upload a file containing multiple sequences. Sequences should be in fasta format.

Absibling

Highly variable region

For B-cell antibody, the most lineage-defining element is usually the CDR3 region. Given the mechanism of B-cell antibody maturation, CDR3 region is highly variable across lineages in terms of sequence and length. By providing the amino acid sequence CDR3 region, users instruct Absibling to focus on relevant antibody sequences within closely related lineages. In this analysis, there is no need to provide full-length antibody sequence that is often highly sensitive R&D secret. Due to the short length of CDR3 region, it might be helpful to include ~5 flanking amino acids on both sides to ensure sensitivity of search.

Conservation threshold

This section defines the extent of conservation within variable region between query and searched target sequences. When flanking peptids are included, the conservation applies to entire length of query sequence including flanking peptids if provided.

Primer with MID tag

Provide the primer sequence that is tagged with molecular identifier (MID). It should contain only sequence complementary to the corresponding antibody coding region (excluding the MID), for example the "IgG-C" primer in the figure below. This information helps Absibling to extract MID in order to track PCR amplicons derived from the same RNA molecule.

Variable gene identity

Specify the identity of the variable gene germline. If not sure, analyze your antibody nucleotide sequence using  Abgermline. It is crucial that germline identity is identified using the same database as we are using.

NGS data

Drag and drop Fastq data derived from Illumina pair-end sequencing. Accepted file types include fastq (*.fastq) and compressed fastq file (*.gz). The sequenced library should be prepared following the method illustrated below or similar. To be compatible, The constant region primer used for reverse-transcription should be tagged with molecular identifier (MID). The relative positions of P7 and P5 primers and sequence of IgG-V do not impact the analysis.

Support level tag

When a sibling sequence is supported by more than one RNA molecule and at least one of the RNA molecule is represented by more than one read, this sibling is labeled as 'mMmR' (see below figure). When a sibling sequence is supported by one RNA molecule which is represented by more than one read, the sibling is labeled as 'sMmR'. Similarly, the scenarios for 'mMsR' and 'sMsR' are illustrated in the figure below.



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Gaithersburg, MD 20878

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Ⓒ 2020 Eliteimmune Corporation. All Rights Reserved
704 Quince Orchard Road, Suite 215
Gaithersburg, MD 20878

External sites:  COVID-19  NCBI  IgBlast  IMGT  PDB