No debe girar la jeringa y el vial al revés, el vial debe estar en posición vertical. Introducir la aguja

Illumina reads from the African buffalo genome project were mapped to the cattle ARS- UCDv0.1 IGL scaffold 1160 and subsequently de novo assembled in SPAdes into the entire IGL locus and as targeted assembly of individual gene segments. As anticipated, complete assembly was impossible due to the highly repetitive nature of the locus and the short sequence length of the Illumina reads. Genome reads that mapped to the ARS-UCDv0.1 scaffold assembled into a heavily fragmented assembly; a total of 42 contigs were assembled, the largest being 7488 bp with an N50 of 4410 bp. Coverage across the assembly was on average 25 x but with highest coverage in the intronic regions. The assembly spanned 180.7 kb but contained only 17 IGLV gene segments and 3 IGLJ-IGLC cassettes. Due to the difficulties in assembling the complete African buffalo IGL, a dot plot assembly comparing the African buffalo IGL to the cattle IGL was highly fragmented and did not show a clear relationship between the two assemblies.

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The IGL gene segments in the African buffalo were then individually de novo assembled in SPAdes with reads mapped to individual cattle IGLV in the ARS-UCDv0.1. A total of 58

IGLV were assembled; 36 IGLV were assembled from reads with 4% alignment error and a

further 12 IGLV were found when alignment error was increased to 10%. Concatenation of the assembled IGLV revealed a total length of 123 kb, An N50 of 61.8 kb with a GC content of 50.96. The average coverage of the IGLV was 10.2x and the contig containing the IGLJ-

IGLC cassettes was 6x (Table 4.1).

African buffalo genome sequencing reads did not map to the remaining six scaffolds containing IGLV in the ARS-UCDv0.1 including to the IGLV gene segments contained within these scaffolds. Subsequent assembly of the whole locus or of individual gene segments contained on these scaffolds was not possible. This could be due to the IGL in African buffalo being less expanded in cattle and therefore missing these gene segments. Alternatively, the highly repetitive nature of these gene segments meant that either mapping of the short Illumina reads to this region was difficult or the locus may have been artificially expanded in the cattle ARS-UCDv0.1.

Figure 4.4: Schematic organisation of the African buffalo IGL de novo assembled in SPAdes using paired end reads that mapped to the cattle ARS-UCDv0.1 IGL scaffold 1160. The assembled region spans 181 kb, consisting of 42 contigs indicated by the black projection lines below the annotation. Red lines indicate an IGLV gene segment, orange lines indicate

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Table 4.1: Mapping statistics of IGLV and IGKV assembled in African buffalo with whole genome reads mapped to the cattle IGL and IGK loci in the ARSUCDv0.1. A total of 58 IGLV and 20 IGKV were assembled which formed seven and two phylogenetic sub-groups respectively, designated by lower case letters.

Buffalo Gene name Contigs Coverage Assembly GC Contig length Mismatch

IGLVa-1(I) 1 2.2 56.2 780 10 IGLVa-2(I) 1 50 54.9 650 IGLVa-3(I) 1 1.7 60.6 536 10 IGLVa-4(I) 2 3.3 56.7 811 IGLVa-5(I) 1 11.2 57.6 968 IGLVa-6(I) 1 7.2 57.3 1039 IGLVa-7(I) 1 24.5 58.4 1146 IGLVa-8(I) 1 5 59.5 993 IGLVa-9(I) 1 10.2 57.5 602 IGLVa-10(I) 2 0.5 59.5 545 10 IGLVa-11(I) 1 9.1 57.1 1061 IGLVa-12(I) 1 27.2 56.6 947 IGLVa-13(I) 1 11.9 56.2 582 IGLVb-1 1 21.6 61.2 1127 10 IGLVb-2 1 2.1 60.3 966 10 IGLVb-3 1 3 58.8 600 IGLVb-4 1 0.6 60.8 600 IGLVb-5 1 3.1 59.1 745 10 IGLVb-6 3 2 59.2 1201 IGLVb-7 1 0.9 58.5 701 IGLVb-8 1 2.2 61.5 774 IGLVb-9 1 2.9 58.7 572 IGLVb-10 1 10.3 57.3 1062 IGLVb-11 1 8.1 60.5 868 IGLVc-1 1 13.4 49.8 1214 IGLVc-2 1 10 54.4 1148 IGLVc-3 1 13.9 57.3 1300 IGLVc-4 2 8.3 56 1085 IGLVd-1 1 20.2 63.8 1037 IGLVd-2 1 10.8 63.6 965 IGLVd-3 1 28.5 61.3 1229 10 IGLVd-4 1 11 60.6 1189 IGLVe-1 1 10.4 65.2 1384 IGLVe-2 1 7.9 62.5 1234 IGLVe-3 1 8.7 64 1182 IGLVe-4 1 11.3 62.9 894 IGLVe-5 1 10 61 1112 10 IGLVf-1 1 6.6 60.8 926 IGLVf-2 2 20.5 58.9 754 IGLVf-3 1 8.4 61 1132 IGLVf-4 1 12.8 59 1174 IGLVf-5 1 24.2 52.6 1467 IGLVf-6 1 3 54.5 765 10 IGLVf-7 1 15.9 52.9 1449 IGLVf-8 1 4.9 53.2 847 IGLVf-9 1 4 56 489 IGLVf-10 1 2 54.7 737 10 IGLVf-11 1 2.4 52 665 10 IGLVg-1 1 2.5 57.8 384 IGLVg-2 1 13.6 57.7 1111 IGLVg-3 1 10.8 56.3 837 IGLVg-4 1 3.3 58.1 640 IGLVg-5 1 9 58.6 1215 IGLVg-6 1 13.5 60.1 781 IGLVg-7 1 11.8 60 1036 10 IGLVg-8 1 15.8 59.5 1108 IGLVg-9 1 16.2 58.4 969 IGLVg-10 1 7.4 57.2 749 IGKVa-1 1 15.5 52.4 1027 10 IGKVa-2 1 17.4 48.4 1076 10 IGKVa-3 1 15.2 43.7 616 10 IGKVa-4 1 18.6 50.2 944 10 IGKVa-5 1 10.4 56.9 695 10 IGKVa-6 1 18.4 49 1061 10 IGKVa-7 1 16.3 52.9 1056 10 IGKVb-1 1 10.6 48.6 1315 10 IGKVb-2 1 7.2 46.4 1242 10 IGKVb-3 1 5.2 46.1 1212 10 IGKVb-4 1 10.7 45.6 1177 10 IGKVb-5 1 6.1 48.2 1341 10 IGKVb-6 1 6.2 47.8 1141 10 IGKVb-7 1 18.2 47.7 1331 10 IGKVb-8 1 9.8 48.8 1108 10 IGKVb-9 2 8.13 46.8 1073 10 IGKVb-10 1 13 47.3 1312 10 IGKVb-11 1 20.2 46 1366 10 IGKVb-12 1 20.3 47.3 1235 10 IGKVb-13 1 17.4 47.4 815 10

144 4.3.5 Assembly of the African buffalo IGK

The African buffalo IGK was assembled by mapping genome reads to the cattle ARS- UCDv0.1 IGK loci and de novo assembling both the entire locus and individual gene segments in SPAdes. The assembly of the entire IGK locus was fragmented on 38 contigs, the largest contig being 10.7 kb, with an N50 of 5350 bp. The IGK contained a truncated

IGKC gene segment, missing the first 9 bp, and 14 IGKV gene segments (Figure 4.5).

Average coverage across the locus was 15.74 x. A total of 20 IGKV were found when individually assembled (Table 1), with the corresponding gene segment in the whole locus assembly having a 98-100% nucleotide sequence identity. The one IGKC and four IGKJ were each assembled on a single contig with a 99% nucleotide identity to the corresponding gene segments in the whole locus assembly. Concatenation of the IGK gene segments spans 24.5 kb with an N50 of 1177 and an average coverage of 13.4 x. IGK in African buffalo appears to be the most complete whole immune loci assembly achieved with the short-read Illumina sequences, although it is still heavily disrupted. A dot plot comparison of the African buffalo and cattle ARS-UCDv0.1 IGK reveals heavy fragmentation in the African buffalo assembly across the variable region, as is expected, but a nearly contiguous assembly at the 3’ end of the locus.

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Figure 4.5: A dot plot comparison of the ARS-UCDv0.1 IGK locus and the African buffalo IGK de novo assembled as an entire locus with reads mapped to the ARS-UCDv0.1. Red lines indicate an IGKV gene segment, yellow indicates the IGKC and the blue line indicates the cluster if IGKJ; cattle and African buffalo both possess four IGKJ. The IGK in the ARS- UCDv0.1 is contained on a single contig whilst the African buffalo assembly is on 38 contigs, the breaks indicated by the black lines on the right.