Edit I stand corrected. @Daniel Standage correctly pointed out that the below is only true for a single query, or for the sorting of hits within those found by a given query. Otherwise they're sorted in the order they were given.

BLAST results are sorted in descending order by the "Bit score" (column 12). Sorting by E. value would not be quite the same.

i.e. column 11 (E value) will be presented in ascending order (an E. value of 0 is as good as it gets), and as it gets higher, your hit is getting worse. Likewise, bitscores are highest at the top and decrease down the list. A hit with E. value = 0 should have a high score. How high exactly depends on the hit length, so it's common to see many E values with 0.0, but the scores differ. Here's a snippet of one of my blast results:

PVCcif_ATCC43949  PAU_06042014  100.000  24180  0     0   1      24180  2233012  2257191  0.0  43606
PVCcif_ATCC43949  PAU_06042014  78.098   5593   1164  16  2765   8323   3219956  3225521  0.0  4547
PVCcif_ATCC43949  PAU_06042014  77.549   5394   1139  16  2948   8323   2376163  2370824  0.0  4260
PVCcif_ATCC43949  PAU_06042014  74.140   5669   1342  34  8490   14100  3225688  3231290  0.0  3573
PVCcif_ATCC43949  PAU_06042014  76.835   3665   825   9   16507  20150  3234191  3237852  0.0  2764
PVCcif_ATCC43949  PAU_06042014  76.871   2646   571   11  16625  19248  2362597  2359971  0.0  2001
PVCcif_ATCC43949  PAU_06042014  70.733   4131   1005  51  9047   13063  2368869  2364829  0.0  1954
PVCcif_ATCC43949  PAU_06042014  75.040   1891   448   7   8500   10383  2370771  2368898  0.0  1272
PVCcif_ATCC43949  PAU_06042014  78.986   1361   269   7   22766  24119  890051   891401   0.0  1150
PVCcif_ATCC43949  PAU_06042014  77.819   1082   217   5   536    1610   3217983  3219048  0.0  868

As you can see, they all have E values of 0.0, but they differ quite a lot in score, this is because of the length of the alignment, and the different percentage IDs.

Edit I stand corrected. @Daniel Standage correctly pointed out that the below is only true for a single query, or for the sorting of hits within those found by a given query. Otherwise they're sorted in the order they were given.

BLAST results are sorted in descending order by the "Bit score" (column 12). Sorting by E. value would not be quite the same.

i.e. column 11 (E value) will be presented in ascending order (an E. value of 0 is as good as it gets), and as it gets higher, your hit is getting worse. Likewise, bitscores are highest at the top and decrease down the list. A hit with E. value = 0 should have a high score. How high exactly depends on the hit length, so it's common to see many E values with 0.0, but the scores differ. Here's a snippet of one of my blast results:

PVCcif_ATCC43949  PAU_06042014  100.000  24180  0     0   1      24180  2233012  2257191  0.0  43606
PVCcif_ATCC43949  PAU_06042014  78.098   5593   1164  16  2765   8323   3219956  3225521  0.0  4547
PVCcif_ATCC43949  PAU_06042014  77.549   5394   1139  16  2948   8323   2376163  2370824  0.0  4260
PVCcif_ATCC43949  PAU_06042014  74.140   5669   1342  34  8490   14100  3225688  3231290  0.0  3573
PVCcif_ATCC43949  PAU_06042014  76.835   3665   825   9   16507  20150  3234191  3237852  0.0  2764
PVCcif_ATCC43949  PAU_06042014  76.871   2646   571   11  16625  19248  2362597  2359971  0.0  2001
PVCcif_ATCC43949  PAU_06042014  70.733   4131   1005  51  9047   13063  2368869  2364829  0.0  1954
PVCcif_ATCC43949  PAU_06042014  75.040   1891   448   7   8500   10383  2370771  2368898  0.0  1272
PVCcif_ATCC43949  PAU_06042014  78.986   1361   269   7   22766  24119  890051   891401   0.0  1150
PVCcif_ATCC43949  PAU_06042014  77.819   1082   217   5   536    1610   3217983  3219048  0.0  868

As you can see, they all have E values of 0.0, but they differ quite a lot in score, this is because of the length of the alignment, and the different percentage IDs.

Actually, by default, BLAST results are sorted in descending order by the "Bit score" (column 12). Sorting by E. value would not be quite the same.

i.e. column 11 (E value) will be presented in ascending order (an E. value of 0 is as good as it gets), and as it gets higher, your hit is getting worse. Likewise, bitscores are highest at the top and decrease down the list. A hit with E. value = 0 should have a high score. How high exactly depends on the hit length, so it's common to see many E values with 0.0, but the scores differ. Here's a snippet of one of my blast results:

PVCcif_ATCC43949  PAU_06042014  100.000  24180  0     0   1      24180  2233012  2257191  0.0  43606
PVCcif_ATCC43949  PAU_06042014  78.098   5593   1164  16  2765   8323   3219956  3225521  0.0  4547
PVCcif_ATCC43949  PAU_06042014  77.549   5394   1139  16  2948   8323   2376163  2370824  0.0  4260
PVCcif_ATCC43949  PAU_06042014  74.140   5669   1342  34  8490   14100  3225688  3231290  0.0  3573
PVCcif_ATCC43949  PAU_06042014  76.835   3665   825   9   16507  20150  3234191  3237852  0.0  2764
PVCcif_ATCC43949  PAU_06042014  76.871   2646   571   11  16625  19248  2362597  2359971  0.0  2001
PVCcif_ATCC43949  PAU_06042014  70.733   4131   1005  51  9047   13063  2368869  2364829  0.0  1954
PVCcif_ATCC43949  PAU_06042014  75.040   1891   448   7   8500   10383  2370771  2368898  0.0  1272
PVCcif_ATCC43949  PAU_06042014  78.986   1361   269   7   22766  24119  890051   891401   0.0  1150
PVCcif_ATCC43949  PAU_06042014  77.819   1082   217   5   536    1610   3217983  3219048  0.0  868

As you can see, they all have E values of 0.0, but they differ quite a lot in score, this is because of the length of the alignment, and the different percentage IDs.

The reason for your appended sequences messing up the ordering, I would suspect, is that in your second sets of sequences, there were some better hits than in your first sets of sequences. Thus, they were sorted by the Bitscore and consequently your hits became interspersed.

Edit I stand corrected. @Daniel Standage correctly pointed out that the below is only true for a single query, or for the sorting of hits within those found by a given query. Otherwise they're sorted in the order they were given.

BLAST results are sorted in descending order by the "Bit score" (column 12). Sorting by E. value would not be quite the same.

i.e. column 11 (E value) will be presented in ascending order (an E. value of 0 is as good as it gets), and as it gets higher, your hit is getting worse. Likewise, bitscores are highest at the top and decrease down the list. A hit with E. value = 0 should have a high score. How high exactly depends on the hit length, so it's common to see many E values with 0.0, but the scores differ. Here's a snippet of one of my blast results:

PVCcif_ATCC43949  PAU_06042014  100.000  24180  0     0   1      24180  2233012  2257191  0.0  43606
PVCcif_ATCC43949  PAU_06042014  78.098   5593   1164  16  2765   8323   3219956  3225521  0.0  4547
PVCcif_ATCC43949  PAU_06042014  77.549   5394   1139  16  2948   8323   2376163  2370824  0.0  4260
PVCcif_ATCC43949  PAU_06042014  74.140   5669   1342  34  8490   14100  3225688  3231290  0.0  3573
PVCcif_ATCC43949  PAU_06042014  76.835   3665   825   9   16507  20150  3234191  3237852  0.0  2764
PVCcif_ATCC43949  PAU_06042014  76.871   2646   571   11  16625  19248  2362597  2359971  0.0  2001
PVCcif_ATCC43949  PAU_06042014  70.733   4131   1005  51  9047   13063  2368869  2364829  0.0  1954
PVCcif_ATCC43949  PAU_06042014  75.040   1891   448   7   8500   10383  2370771  2368898  0.0  1272
PVCcif_ATCC43949  PAU_06042014  78.986   1361   269   7   22766  24119  890051   891401   0.0  1150
PVCcif_ATCC43949  PAU_06042014  77.819   1082   217   5   536    1610   3217983  3219048  0.0  868

As you can see, they all have E values of 0.0, but they differ quite a lot in score, this is because of the length of the alignment, and the different percentage IDs.