...

If the alignment was generated in Partek® Flow®, the genome assembly will be displayed as text on the top of the page (Figure 1), you do not have the option to change the reference.

If the bam file is imported, you need to select the assembly with which the reads were aligned to, and which annotation model file you will use to quantify from the drop-down menus (Figure 2).

In the Quantification options section, when the Strict paired-end compatibility check button is selected, paired end reads will be considered compatible with a transcript only if both ends are compatible with the transcript. If it is not selected, reads with only one end have alignment that is compatible with the transcript will also be counted for the transcript .

...

• No: Reads will be included in the calculation as long as they map to exonic regions, regardless of the direction.

• Auto-detect: The first 200,000 reads will be used to examine the strand compatibility with the transcripts. Two percentages are calculated: (1) the percentage of reads whose first-in-pair is the same strand as the transcript and second-in-pair is the opposite strand to transcript, (2) the percentage of reads whose first-in-pair is the opposite strand to transcript and second-in-pair is the same strand as the transcript. If the 1st percentage is higher than 75%, the Forward-Reverse option will be used. If the 2nd  percentage is higher than 75%, the Reverse-Forward option will be used. If neither of the percentages exceed 75%, No option will be used.

• Forward – Reverse: this option is equivalent to the --fr-secondstrand option in Cufflinks [1]. First-in-pair is the same strand as the transcript, second-in-pair is the opposite strand to the transcript.

• Reserve – Forward: this option is equivalent to --fr-firststrand option in Cufflinks. First-in-pair is the opposite strand to the transcript, second-in-pair is the same strand as the transcript. The Illumina TruSeq Stranded library prep kit is an example of this configuration.

• Forward – Forward: Both ends of the read are matching the strand of the transcript. Generally colorspace data generated from SOLiD technology would follow this format

...

Depending on the annotation file, the output could be one or two data nodes. If the annotation file only contains one level of information, e.g. miRNA annotation file, you will only get one output data node. On the other hand, if the annotation file contains gene level and transcript level information, such as those from the Ensembl database, both gene and transcript level data nodes will be generated. If two nodes are generated, the Task report will also contain two tabs, reporting quantification results from each node. Each report has two tables. The first one is a summary table displaying the coverage information for each sample quantified against the specified transcriptome annotation (Figure 4).

The second table contains feature distribution information on each sample and across all the samples, number of features in the annotation model is displayed on the table title (Figure 5).

The bar chart displaying the distribution of raw read counts is helpful in assessing the expression level distribution within each sample. The X-axis is the read count range, Y axis is the number of features within the range, each bar is a sample. Hovering your mouse over the bar displays the following information (Figure 6):

• Sample name

• Range of read counts, “[ “represent inclusive, “)” represent exclusive, e.g. [0,0] means 0 read counts; (0,10] means the range is greater than 0 count but less than and equal to 10 counts.

• Number of features within the read count range

• Percentage of the features within the read count range

The coverage breakdown bar chart is a graphical representation of the reads summary table for each sample (Figure 7)

In the box-whisker plot, each box is a sample on X-axis, the box represents 25th and 75th percentile, the whiskers represent 10th and 90th percentile, Y-axis represents the read counts, when you hover over each box, detailed sample information is displayed (Figure 8).

• Sample name

• Range of read counts, “[ “represent inclusive, “)” represent exclusive

• Number of features within the read count range in the sample

...

• Novel transcript : this option does not require any annotation reference, it will do de novo assembly to reconstruct transcripts and estimate their abundance

• Annotation transcript : this option requires an annotation model to quantify the aligned reads to known transcripts based on the annotation file.

• Novel transcript with annotation as guide: this option requires an annotation file to quantify the aligned reads to known transcripts as well as assemble aligned reads to novel transcripts. The results include all transcripts in the annotation file plus any novel transcripts that are assembled.

...

• Min coverage: will filter out any features (sequence names) that have fewer reads across all samples than the value specified

• Strict paired-end compatibility: this only affects paired end data. When it is checked, only reads that have two ends aligned to the same feature will be counted. Otherwise, reads will still be counted as exonic compatible reads even if the mate is not compatible with the feature

...