PMC

High-Throughput Technologies to Generate, Propagate, and Map Insertions in the Indexed Mutant Library.
Individual transformants were robotically picked into a collection of arrays of 384 colonies on agar and were propagated while insertion sites were being mapped and confirmed. Once the coordinates of high-confidence mutants were identified, these mutants were then arrayed into a final collection that was cryopreserved and is available for distribution.

The Density of Confirmed Insertions Differs Between Gene Features and Intergenic Regions.
The data represent 265 intergenic insertions and 1855 insertions into genes. Categories with insertion densities statistically significantly different from the intergenic density are marked with asterisks (P values < 0.001). The densities are adjusted to compensate for differences in the density of uniquely mappable insertion positions between features (see Methods and Supplemental Methods).

Plate and Colony Coordinates of Each Flanking Sequence Were Separately Determined.
(A) To determine plate coordinates, the mutants were pooled by plate into 53 plate-pools. We combinatorially distributed the plate-pools into 15 plate-super-pools and obtained the flanking sequences in each plate-super-pool by ChlaMmeSeq. We then determined the flanking sequences in each plate by deconvolving the plate-super-pool data. We employed a similar process to identify the flanking sequences present at each colony position. This process revealed the plate and colony coordinates for each flanking sequence.
(B) and (C) The patterns of presence and absence of pools in super-pools are shown for super-pooling by plate (B) and by colony (C).
(D) Most flanking sequences were mapped to both a plate and a colony position.
(E) Most flanking sequence coordinates were identified with no errors. An error indicates that a flanking sequence is present in an unexpected super-pool, or absent from an expected super-pool, but the flanking sequence can still be uniquely mapped to a pool due to the error tolerance of our method.

Screening of Mutants in Genes Encoding the Lipid Droplet Proteome for Defects in Accumulation.
(A) Lipid droplet proteins. In lipid droplets, which store the cell’s triacylglycerol, 433 proteins have been identified by proteomics. Thirty mutants disrupted in 28 of those proteins were found in the library.
(B) The content in LEAP-Seq confirmed mutants disrupted in genes encoding lipid droplet proteins was analyzed. This revealed a defect in accumulation in the lcs2 mutant (shown in red). One plate with seven mutants is presented here; other mutants are presented in Supplemental Figure 13. Lipids extracted from cells (normalized by the same amount of chlorophyll) were loaded into each lane. Each mutant is labeled with the v5.3 ID of the gene the insertion was mapped to. Detailed information is shown in Supplemental Data Set 11.

LCS2 Is Required for Accumulation.
(A) The cassette insertion site in the lcs2 mutant is shown on the LCS2 gene model from the Phytozome v5.3 Chlamydomonas genome. Black solid boxes indicate exons, thin lines indicate introns, and are shown as gray boxes. A fragment of chloroplast DNA is indicated in orange. Primer locations for PCR are shown as arrows. The two red triangles indicate the location of the peptide against which the antibody was raised, which is translated from two neighboring exons.
(B) Examination of the genotypes of lcs2 and complemented lines (comp1-4) by PCR from genomic DNA. PCR primers are indicated to the left of the panel.
(C) Use of immunoblotting to measure LCS2 protein levels of lcs2 and complemented lines. α-Tubulin was used as a loading control.
(D) Use of to measure content in lcs2 and complemented lines.
(E) To confirm the results, was used to quantify content for CC-4533, lcs2, and complemented lines. Error bars indicate standard deviations from three biological replicates.

Most Insertions in the Collection Are Complex.
(A) Our previous work suggested a model where transforming DNA is fragmented, with multiple cassette fragments and/or exogenous genomic DNA sequences sometimes inserted into the same site in the Chlamydomonas genome (adapted from ).
(B) Only one flanking sequence was assigned to most colonies, and two or more flanking sequences were assigned to some colonies. The plot also shows how many of the colonies have only 5′-side flanking sequences, only 3′, or both 5′ and 3′. Two 5′ or two 3′ ends can be derived from a single insertion of two cassettes in opposite orientations, as well as from two independent insertions.
(C) Data from pairs of flanking sequences mapped to the same colony were used to estimate how often different insertion categories were detected in the collection (see Supplemental Figure 5 and Supplemental Methods).

LEAP-Seq Improves Confidence in Insertion Sites.
(A) LEAP-Seq reads are used to identify cases where there is a short insertion of genomic DNA from another locus (designated “junk” DNA) between the cassette and the flanking genomic DNA. LEAP-Seq reads are paired-end, with each pair consisting of a proximal and distal read (relative to the cassette). Short insertions of junk DNA will typically produce a low percentage of distal reads mapped to the same locus as the proximal read, and the read pairs will span a short distance at this locus.
(B) For each 5′ flanking sequence with at least 10 reads, the percentage of distal reads mapped to the same locus as the proximal read is plotted against the maximum distance spanned by read pairs at that locus. Positive control flanking sequences, indicated by blue circles, are 5′ flanking sequences that have perfectly matching 3′ flanking sequences mapped to the same colony. Some control flanking sequences showed a small fraction of distal reads mapping to distant loci as a result of apparent artifacts (see Supplemental Methods). The dotted line shows the cutoffs for insertions that we consider confirmed by LEAP-Seq.
(C) PCR validation of LEAP-Seq-confirmed insertions. Results show that 33/44 LEAP-Seq-confirmed insertions were correctly mapped; a further three confirmed insertions could not be checked due to PCR failure (Supplemental Figure 9).
(D) PCR validation for non-LEAP-Seq-confirmed insertions. Results show that 4/42 were correctly mapped; seven could not be checked due to PCR failure.

LCS2 Is a Key Factor in Fatty Acid Export from the Chloroplast for Synthesis.
(A) was used to quantify different molecular species of in the lcs2 mutant and complemented lines in three biological replicates. Each species is denoted as N:M, where N is the number of carbon atoms in acyl chains and M is the number of C=C double bonds. In each strain, the average percentage of total represented by each species is indicated by the size of the corresponding circle. Next to each circle, the percentage abundance of that species in each biological replicate is shown relative to the average percentage abundance of that species in CC-4533, as a heat map. A Mann-Whitney U-test was used to test whether the values in the heat map of lcs2 deviate significantly from the values in strains expressing LCS2 (CC-4533 plus the complemented lines) (asterisk indicates false discovery rate corrected P < 0.02).
(B) A model is proposed for the role of LCS2 in synthesis under nitrogen deprivation. Enzymes and processes are italicized. Dashed arrows indicate processes that may involve multiple steps. Two major sources of acyl groups are represented: (1) LCS2 activates de novo synthesized fatty acids exported from the plastid and provides acyl-CoA for synthesis; and (2) degradation of existing membrane lipids at the plastid or endoplasmic reticulum (ER) can provide acyl groups to through LCS2-independent pathways. ACP, acyl carrier protein; FA, fatty acid; FAS, fatty acid synthesis.