Instructions on PCRs to check the insertion site
1. Q: What is the background strain?
A: The strain we used to generated mutants with is CMJ030, also known as CC-4533. Details can be found on the background strain page.
2. Q: What is the sequence of the insertion cassette? What gene is the resistance marker?
A: The cassette we used to generate mutants labeled "LMJ.SG0182.******" was obtained from digestion of a plasmid pMJ13b with MlyI enzyme. The cassette we used to generated mutants labeled "LMJ.RY0402.******" is CIB1. Both cassettes contain a gene that confers resistance to paromomycin.
3. Q: How many insertions per mutant are there on average?
A: By Southern blotting with the coding sequence of the resistance gene as a probe, we found that 18/24 mutants had a single insertion and 6/24 had two.
4. Q: After opening the mutant strain page, where do I find the details about the insertion?
A: Each strain might harbor multiple insertions or multiple insertion junctions derived from a single insertion. Insertion junctions are listed in a table on the bottom of the strain information page. Features associated with each insertion junction can be found after clicking on the corresponding insertion junction. See the "Specific Fields in Mutant and Insertion Junction Pages" section below for details on how to interpret the information.
5. Q: How reliable are the mapping data?
A: We did PCRs to verify the insertion site is indeed disrupted for a number of mutants. For the mutants we analyzed, in 75% of cases, the mapping can be confirmed. We urge you to confirm that your gene of interest is disrupted by using this PCR protocol.
6. Q: How should I interpret the flanking sequence?
A: It was read from the genome side from the cassette outward. Detailed information on strandness of the flanking sequence and how to confirm the flanking sequence can be found in the check-PCR instructions.
7. Q: For my mutant the insertion junction is only known for one side of the cassette. How do I find the junction on the other side?
A: Information on how to confirm the insertion site and how to find the genome-cassette junction on the other side can be found in the check-PCR instructions.
8. Q: Who should we acknowledge when we publish research using the mutants?
A: We recommend this statement in publications: We thank the Chlamydomonas Mutant Library Group at Princeton University, the Carnegie Institution for Science, and the Chlamydomonas Resource Center at the University of Minnesota for providing the indexed Chlamydomonas insertional mutant(s).
Insertion junctions: Each mutant may have one or multiple sequenced insertion junctions, which may be two sides of the same insertion, or multiple independent cassette insertions in one mutant. Two sides of the same insertion will usually be 5’ and 3’, but may sometimes both be 5’ or both be 3’, if the insertion consisted of two cassettes in reverse orientations. Sometimes there is a genomic DNA fragment on one side of the insertion - approximately 25% insertion junction positions are incorrect due to this and other factors. This can also result in two sides of the same insertion being mapped to distant locations.
Locus name and Common name: The ID and common name of the gene containing the insertion. If the insertion junction is inside two overlapping genes, they’re separated with &. Some genes have no common names.
Feature: Which feature of the gene the insertion is in. For genes with multiple splice variants, this wasn’t determined. For overlapping genes, there are two &-separated values. For insertions precisely between two features, the two features are separated with /.
Side of cassette: Which side of the cassette this insertion junction is on. Insertions should have two sides, although often only one has been sequenced; the two sides will usually be 5’ and 3’, but may sometimes both be 5’ or both be 3’, if the insertion consisted of two cassettes in reverse orientations.
Location: The genomic base immediately before the cassette insertion, 1-based.
Cassette orientation: ‘+’ if the insertion cassette’s resistance gene is transcribed in the + chromosome direction, ‘-’ otherwise.
Orientation: ‘sense’ if the insertion cassette’s resistance gene is transcribed in the same direction as the gene, ‘antisense’ otherwise. For overlapping genes, there are two &-separated values.
Flanking sequence: The genomic sequence immediately flanking the cassette, read from the cassette junction outwards (may match the genome sequence or be a reverse-complement; may have 1bp mismatch compared to reference genome sequence.)
LEAP-Seq_confirmed_distance: The longest distance between the proximal and distal LEAP-Seq reads for this insertion junction that mapped to the same locus. The longer the distance, the higher the confidence that this insertion location is correct rather than being an artifact due to a genomic DNA fragment inserted with the cassette.
LEAP-Seq_percent_matching_reads: Percent of matching reads - see next two fields for what those are. The higher the percentage, the higher the confidence that this insertion location is correct.
LEAP-Seq_N_matching_reads: Number of LEAP-Seq read pairs in which the proximal and distal reads mapped to the same locus.
LEAP-Seq_N_non-matching_reads: Number of LEAP-Seq read pairs in which the proximal and distal reads mapped to different genomic regions. This can be due to a genomic DNA fragment inserted with the cassette, or to LEAP-Seq artifacts or PCR/sequencing errors.