H06347
SUPPLEMENTARY INFORMATION
Table 1: P. falciparum chromosome 12 YAC reads. Table 1 presents a compendium of
shotgun sequence data derived from the minimum tiling path composed of 21 P.
falciparum chromosome 12 YACs. Shotgun sequence coverage of the YACs varied
considerably, with a range of 0.5- to 9.7-fold YAC coverage (whereas in our Letter, we
presented “chromosome 12 coverage”). Only good quality (phred 2 score of 20 or greater)
bases are presented. In our Letter, we counted only those reads and bases that are part of
the P. falciparum chromosome 12 consensus sequence. Here, we are presenting all of the
good quality reads and bases whether or not they are part of the consensus sequence. For
example, none of the strain B8 reads and bases are part of the consensus sequence. Thus,
there are more reads and bases presented here than are in our Letter. With the exception
of four YACs (341, 293, B8-420, and 25) for which we experimented with high YAC
coverage early in this project, the shotgun sequence coverage of the remaining YACs is
low, as originally planned. We found that YAC 121 is contained entirely within YAC
812. The first column lists the YACs that were shotgun sequenced, reading top-to-bottom
as left-to-right 1. The second column gives the number of shotgun sequence reads for
each YAC. The third column presents the calculation of YAC shotgun sequence coverage
for each YAC: the total number of good quality bases for each YAC divided by the size
of each YAC. The fourth column lists the size of each YAC 1. In Table 1, there is a total
of 19,112 YAC reads with a total of 7,921,348 good quality bases.
YAC
number
YAC
YAC size
of reads coverage (kb)
--------------------------------------------1262
206
2.0
40 kb
341
2182
5.7
170 kb
B8-95
270
1.2
90 kb
492
656
1.1
215 kb
357
193
0.5
135 kb
538
445
0.9
215 kb
724
1020
2.5
165 kb
1122
260
0.8
120 kb
69
409
1.0
165 kb
812
1976
3.0
175 kb
121
159
1.1
50 kb
293
2024
6.3
140 kb
1383
128
0.7
80 kb
B8-628
313
0.6
170 kb
588
273
1.1
90 kb
181
1894
4.0
220 kb
336
350
0.8
150 kb
312
120
1.1
40 kb
B8-420
3735
9.7 170 kb
614
25
995
1504
5.1
8.4
80 kb
90 kb
Table 2. P. falciparum chromosome 12 reads. Table 2 presents a summary of our P.
falciparum chromosome 12 sequence reads. Only good quality (phred 2 score of 20 or
greater) bases are presented. The top half of the table lists the data from shotgun
sequencing chromosome 12. The first column lists the vector (M13 phage or pUC
plasmid) and type of dye chemistry (dye-primer, dye-terminator, and BigDye-terminator)
used for detection on polyacrylamide gels. The second column gives the number of reads.
The third column presents the calculation of raw chromosome 12 coverage for each
category: the total number of good quality bases divided by the size of P. falciparum
chromosome 12. The lower half of the table gives the number of reads accumulated
during the finishing process. In the first column, these reads are divided into groups based
upon the sequencing template: PCR products (sequenced in whole or in part for physical
gap filling, sequencing the complementary strand, or validation reaction product),
existing M13 or pUC templates employed for primer-extension sequencing, and existing
M13 templates for PCR to synthesize and sequence the complementary strand ("M13
reverses"). All finishing sequence reactions were conducted with BigDye-terminator
chemistry. Whereas in our Letter we included only those reads/bases that support the
chromosome 12 consensus sequence, here we are including all of our data. Therefore, the
numbers in Table 2 are higher than the numbers in our Letter. At the beginning of this
large-scale sequencing project, we achieved a combined total of 4.4-fold chromosome 12
coverage with dye-primer and dye-terminator chemistries. For the M13-based reads, the
average good quality read lengths were 342 bases (b) for the dye-primer chemistry and
412 b for the dye-terminator chemistry. A comparison of these two numbers explains
why we undertook such limited sequencing with dye-primer chemistry. With BigDyeterminator chemistry, the average good quality read length in the M13-based vector was
460 b. This considerable improvement in good quality read length explains why we
switched from dye-terminator chemistry to BigDye-terminator chemistry as soon as
BigDye-terminator chemistry became available commercially. Employing BigDyeterminator chemistry, we achieved 6.89-fold chromosome 12 coverage. Thus, in total, we
achieved 8.7-fold shotgun sequence coverage of P. falciparum chromosome 12.
However, the P. falciparum chromosome 12 preparations were approximately 80% pure
3
. Therefore, unsurprisingly, roughly 20% of our P. falciparum chromosome 12 shotgun
reads did not assemble into chromosome 12. These reads are presumptive contaminants
derived from other P. falciparum chromosomes. We imported the equivalent nonassembled reads from our colleagues at the Wellcome Trust Sanger Institute and the
Institute for Genomic Research. Using a stringent standard, we conducted our matching
procedure with those imported reads and our bins. As the result of this matching process,
we found an additional 7,007 shotgun sequence reads ("imported" in Table 2). As we did
not have phred scores for the bases, we could not calculate coverage for these reads.
Shotgun
Number of reads Coverage
M13 dye-primer
1,857
0.17
M13 dye-terminator 12,389
1.64
M13 BigDye-term
41,898
6.89
pUC dye-term
imported
13,388
7,007
2.58
---
Finishing
PCR reads
primer-extension
M13 reverses
303
6,901
205
0.03
1.02
0.04
1. Rubio, J. P., Thompson, J. K. & Cowman, A. F. The var genes of Plasmodium
falciparum are located in the subtelomeric region of most chromosomes. Embo J 15,
4069-77. (1996).
2. Ewing, B., Hillier, L., Wendl, M. C. & Green, P. Base-calling of automated sequencer
traces using phred. I. Accuracy assessment. Genome Res 8, 175-85. (1998).
3. Su, X. Z. & Wellems, T. E. Plasmodium falciparum: assignment of microsatellite
markers to chromosomes by PFG-PCR. Exp Parasitol 91, 367-9. (1999).