QC Metrics for immune repertoire results

In the Sample QC tab, select a QC metric from the Views list to see a graphical representation of QC metrics.

Read classification

After the first stage of data processing, raw sequencing reads are classified and proportionally represented in a stacked bar plot. Actual read counts for each classification are listed below the figure in the results report.

Read classification

Description

Example

Off-target/low-quality (dark gray)

Reads that are of low quality or represent the product of an off-target amplification.

Unproductive (gray)

Reads that have uncorrectable sequencing or PCR errors that lead the rearrangement to have out-of-frame variable and joining genes or a premature stop codon.

Rescued productive (light blue)

Reads that have an in-frame variable and joining gene, and no stop codons after InDel error correction.

Productive (blue)

Reads that have an in-frame variable and joining gene, and no stop codons.

Proportion of full length, quality trimmed and reads lacking P1-key, by read classification

Stacked bar plot indicating the frequency of quality trimming for reads classified as productive, rescued productive, unproductive, and off-target/low-quality. Full length reads categorized as low quality/off-target likely represent off-target amplifications.

Color

Description

Example

Red

Quality trimmed reads lacking an identifiable P1-key.

Yellow

Quality trimmed reads having an identified P1-key.

Green

Full length reads having an identified P1-key, and no quality trimming.

Base composition of overcalled and undercalled homopolymers

Stacked bar plot indicating the nucleotide composition of overcalled bases (base insertion sequencing errors) and undercalled bases (base deletion sequencing errors). Highly skewed nucleotide composition can indicate lower quality sequencing or low library diversity.

Color

Description

Example

Yellow

Guanidine (G)

Blue

Cytosine (C)

Ted

Thymidine (T)

Green

Adenosine (A)

Downsampling analysis

Downsampling is achieved by repeating clone identification and measurement of repertoire features using 10 K, 50 K, 250 K, 500 K, 750 K, 1 M, 1.5 M, 2 M, and 5 M randomly selected productive and rescued productive reads, contingent on sequencing depth. The graphs show the effect of sequencing depth on select repertoire features: number of clones detected, lineages detected, clone & lineage evenness, and the clone & lineage Shannon diversity. Values for these repertoire metrics that are displayed in this plot are provided in the metrics file.

Clone summary and lineage summary files that are derived from downsampled data are provided in the 'downsampling' subdirectory of the zipped results download file. If insufficient reads are available for a particular downsampling depth, the corresponding fields are assigned a 'NA' value in the metrics file.

Color

Description

Example

Green

Clones & Lineages detected

Blue

Clone & Lineage Shannon diversity

Orange

Clone & Lineage Evenness

QC metrics

The QC metrics include the read classification counts and strand QC metrics.

Category

Description

Read classification

Total productive reads

Productive + rescued productive reads.

Productive reads

Reads having an in-frame variable and joining gene, and no stop codons.

Rescued productive reads

Reads having an in-frame variable and joining gene, and no stop codons after InDel error correction.

Unproductive reads

Reads that have uncorrectable sequencing or PCR errors that lead the rearrangement to have out-of-frame variable and joining genes or a premature stop codon.

Off-target/ low‑quality

Reads which are of low quality or represent the product of an off-target amplification.

Strand QC metrics

Plus strand (v‑side) read counts

Number of sequence read counts from the plus (+) strand.

Minus strand (c‑side) read counts

Number of sequence read counts from the minus (–) strand.

Plus strand CDR3 avg PHRED

Average PHRED score for plus (+) strand reads.

Minus strand CDR3 avg PHRED

Average PHRED score for minus (–) strand reads.